Compositions comprising complement receptor type 1 molecules having carbohydrate structures that are selectin ligands

ABSTRACT

The present invention provides compositions comprising at least one complement moiety and at least one carbohydrate moiety, and methods of producing such compositions. In particular, the compositions of the invention comprise complement proteins related to the complement receptor type 1, and further comprise ligands for intercellular molecules, such as selectins. In a preferred embodiment, the compositions comprise a complement-related protein in combination with the Lewis X antigen or the sialyl Lewis X antigen. The compositions of the invention have use in the diagnosis or therapy of disorders involving complement activity and inflammation. Pharmaceutical compositions are also provided for treating or reducing inflammation mediated by inappropriate complement activity and intercellular adhesion.

This is a continuation of international application No. PCT/US94/05285,filed May 12, 1994 and designating the United States, which is a CIP ofU.S. application Ser. No. 08/061,982, filed May 17, 1993 and nowabandoned.

1. FIELD OF THE INVENTION

In its broadest aspect, the present invention provides compositionscomprising at least one complement moiety and at least one carbohydratemoiety, and methods of producing such compositions. In particular, thecompositions of the invention comprise complement proteins related tothe complement receptor type 1, and further comprise ligands forintercellular adhesion molecules, such as selectins. In a preferredembodiment, the compositions comprise a complement receptor type 1, orfragment or derivative thereof, in combination with the Lewis X antigenor the sialyl Lewis X antigen. The compositions of the invention haveuse in the diagnosis or therapy of disorders involving complementactivity and inflammation. Pharmaceutical compositions are also providedfor treating or reducing inflammation mediated by inappropriatecomplement activity and intercellular adhesion.

2. BACKGROUND OF THE INVENTION

2.1. THE COMPLEMENT SYSTEM

The complement system is a group of proteins that constitute about 10percent of the globulins in the normal serum of humans (Hood, L. E., etal., 1984, Immunology, 2d Ed., The Benjamin/Cummings Publishing Co.,Menlo Park, Calif., p. 339). Complement (C) plays an important role inthe mediation of immune and allergic reactions (Rapp, H. J. and Borsos,T, 1970, Molecular Basis of Complement Action, Appleton-Century-Crofts(Meredity), New York). The activation of complement components leads tothe generation of a group of factors, including chemotactic peptidesthat mediate the inflammation associated with complement dependentdiseases. The sequential activation of the complement cascade may occurvia the classical pathway involving antigen-antibody complexes, or bythe alternative pathway which involves the recognition of foreignstructures such as, certain cell wall polysaccharides. The activitiesmediated by activated complement proteins include lysis of target cells,chemotaxis, opsonization, stimulation of vascular and other smoothmuscle cells, and functional aberrations such as degranulation of mastcells, increased permeability of small blood vessels, directed migrationof leukocytes, and activation of B lymphocytes and macrophages (Eisen,H. N., 1974, Immunology, Harper & Row Publishers, Inc. Hagerstown, Md.,p. 512).

During proteolytic cascade steps, biologically active peptide fragments,the anaphylatoxins C3a, C4a, and C5a (See WHO Scientific Group, 1977,WHO Tech Rep. Ser. 606:5 and references cited therein), are releasedfrom the third (C3), fourth (C4), and fifth (C5) native complementcomponents (Hugli, T. E., 1981, CRC Crit. Rev. Immunol. 1:321; Bult, H.and Herman, A. G., 1983, Agents Actions 13:405).

2.2. COMPLEMENT RECEPTORS

COMPLEMENT RECEPTOR 1 (CR1). The human C3b/C4b receptor, termed CR1 orCD35, is present on erythrocytes, monocytes/macrophages, granulocytes, Bcells, some T cells, splenic follicular dendritic cells, and glomerularpodocytes (Fearon D. T., 1980, J. Exp. Med. 152:20, Wilson, J. G., etal., 1983, J. Immunol. 131:684; Reynes, M., et al., 1976 N. Engl. J.Med. 295:10; Kazatchkine, M. D., et al., 1982, Clin. Immunol.Immunopathol. 27:210). CR1 specifically binds C3b, C4b and iC3b.

CR1 can inhibit the classical and alternative pathway C3/C5 convertasesand act as a cofactor for the cleavage of C3b and C4b by factor I,indicating that CR1 also has complement regulatory functions in additionto serving as a receptor (Fearon, D. T., 1979, Proc. Natl. Acad. Sci.U.S.A. 76:5867; Iida, K. I. and Nussenzweig, V., 1981, J. Exp. Med.153:1138). In the alternative pathway of complement activation, thebimolecular complex C3b,Bb is a C3 enzyme (convertase). CR1 (and factorH, at higher concentrations) can bind to C3b and can also promote thedissociation of C3b,Bb. Furthermore, formation of C3b,CR1 (and C3b,H)renders C3b susceptible to irreversible proteolytic inactivation byfactor I, resulting in the formation of inactivated C3b (iC3b). In theclassical pathway of complement activation, the complex C4b,2a is the C3convertase.

CR1 (and C4 binding protein, C4bp, at higher concentrations) can bind toC4b, and can also promote the dissociation of C4b,2a. The bindingrenders C4b susceptible to irreversible proteolytic inactivation byfactor I through cleavage to C4c and C4d (inactivated complementproteins).

CR1 has been shown to have homology to complement receptor type 2 (CR2)(Weis, J.J., et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:5639-5643).CR1 is a glycoprotein comprising multiple short consensus repeats (SCRs)arranged in 4 long homologous repeats (LHRs). The most C-terminal LHRcalled LHR-D is followed by 2 additional SCRs, a transmembrane regionand a cytoplasmic region (Klickstein, et al., 1987, J. Exp. Med.,165:1095; Klickstein, et al. 1988, J. Exp. Med., 168:1699-1717).Erythrocyte CR1 appears to be involved in the removal of circulatingimmune complexes in autoimmune patients and its levels may correlatewith the development of AIDS (Inada, et al., 1986, AIDS Res. 2:235;Inada, et al., 1989, Ann. Rheu. Dis. 4:287).

Four allotypic forms of CR1 have been found, differing by increments of40,000-50,000 daltons molecular weight. The two most common forms, the Fand S allotypes, also termed the A and B allotypes, have molecularweights of 250,000 and 290,000 daltons respectively, (Dykman, T. R., etal., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1698; Wong, W. W., et al.,1983, J. Clin. Invest. 72:685), and two rarer forms have molecularweights of 210,000 and 290,000 daltons (Dykman, T. R., et al., 1984, J.Exp. Med. 159:691; Dykman, T. R., et al., 1985, J. Immunol. 134:1787).These differences apparently represent variations in the polypeptidechain of CR1, rather than glycosylation state, because they were notabolished by treatment of purified receptor protein with endoglycosidaseF (Wong, W. W., et al., 1983, J. Clin. Invest. 72:685), and they wereobserved when receptor allotypes were biosynthesized in the presence ofthe glycosylation inhibitor tunicamycin (Lublin, D. M., et al., 1986, J.Biol. Chem. 261:5736). All four CR1 allotypes have C3b-binding activity(Dykman, T. R., et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:1698;Wong, W. W., et al., 1983, J. Clin. Invest. 72:685; Dykman, T. R., etal., 1984, J. Exp. Ned., 159:691; Dykman, T. R., et al., 1985, J.Immunol. 134:1787). There are four LHRs in the F (or A) allotype of ˜250kD, termed LHR-A, -B, -C, and -D, respectively, 5′ to 3′ (Wong, et al.,1989, J. Exp. Med. 169:847). While the first two SCRs in LHR-A determineits ability to bind C4b, the corresponding units in LHR-B and -Cdetermine their higher affinities for C3b. The larger S (or B) allotypeof ˜290 kd has a fifth LHR that is a chimera of the 5′ half of LHR-B andthe 3′ half of LHR-A and is predicted to contain a third C3b bindingsite (Wong, et al., 1989, J. Exp. Med. 169:847). The smallest F′ (or C)allotype of CR1 of ˜210 kD, found in increased incidence in patientswith systemic lupus erthematosis (SLE) and associated with patients inmultiple lupus families (Dykman, et al., 1984, J. Exp. Med. 159:691; VanDyne, et al., 1987, Clin. Exp. Immunol. 68:570), may have resulted fromthe deletion of one LHR and may be impaired in its capacity to bindefficiently to immune complexes coated with complement fragments.

A naturally occurring soluble form of CR1 has been identified in theplasma of normal individuals and certain individuals with SLE (Yoon, etal., 1985 J. Immunol. 134:3332-3338). Its structural and functionalcharacteristics are similar to those of erythrocyte (cell surface) CR1,both structurally and functionally. Hourcade, et al. (1988, J. Exp. Med.168:1255-1270) also observed an alternative polyadenylation site in thehuman CR1 transcriptional unit that was predicted to produce a secretedform of CR1 containing the C4b binding domain.

Several soluble fragments of CR1 have also been generated viarecombinant DNA procedures by eliminating the transmembrane region fromthe DNAs being expressed (Fearon, et al., International PatentPublication No. WO89/09220, Oct. 5, 1989; Fearon, et al., InternationalPatent Publication No. WO91/05047, Apr. 18, 1991). The soluble CR1fragments were functionally active, bound C3b and/or C4b anddemonstrated factor I cofactor activity, depending upon the regions theycontained. Such constructs inhibited in vitro the consequences ofcomplement activation such as neutrophil oxidative burst, complementmediated hemolysis, and C3a and C5a production. A soluble constructsCR1/pBSCR1c, also demonstrated in vivo activity in a reversed passiveArthus reaction (Fearon, et al., 1989, supra; Fearon, et al., 1991,supra; Yeh, et al., 1991 supra), suppressed post ischemic myocardialinflammation and necrosis (Fearon, et al., ¹⁹⁸⁹, supra; Fearon, et al.,1991, supra; Weismann, et al., 1990, Science, 249:146-151) and extendedsurvival rates following transplantation (Pruitt and Bollinger, 1991, J.Surg. Res. 50:350; Pruitt, et al., 1991, Transplantation 52:868).[Mulligan et al, 1992, J. Immunol. 148:3086-3092 (injury followingimmune complex deposition). Mulligan, et al., 1992, J. Immunol.148:1479-1485 (protection from neutrophil mediated tissue injury).Lindsay, et al., 1992, Annals of Surg. 216:677. , Hill, et al., 1992, J.Immunol. 149:1722-1728 (tissue ischemia reperfusion injuries)].

CR2. Complement receptor type 2 (CR2, CD21) is a transmembranephosphoprotein consisting of an extracellular domain which is comprisedof 15 or 16 SCRs, a 24 amino acid transmembrane region, and a 34 aminoacid cytoplasmic domain (Moore, et al., 1987, Proc. Natl. Acad. Sci.U.S.A. 84:9194-9198; Weis, et al., 1988, J. Exp. Med. 167:1047-1066).Electron microscopic studies of soluble recombinant CR2 have shown that,like CR1, it is an extended, highly flexible molecule with an estimatedcontour length of 39.6 nanometers by 3.2 nanometers, in which each SCRappears as a ringlet 2.4 nanometers in length (Moore, et al., 1989, J.Biol. Chem. 34:20576-20582).

By means of recombinant DNA experiments with eukaryotic expressionvectors expressing deletion or substitution mutants of CR2 in COS cells,the ligand 30 binding sites of CR2 have been localized to the twoN-terminal SCR's of the molecule (Lowell, et al., 1989, J. Exp. Med.170:1931-1946). Binding by cell surface CR2 of the multivalent forms ofC3 ligands such as iC3b and C3dg causes activation of B-cells (Melchers,et al., 1985, Nature, 317:264-267; Bohnsack, et al., 1988, J. Immunol.141:457-463; Carter, et al., 1988, J. Immunol. 143:1755-1760).

A form of recombinant soluble CR2 has been produced (Moore, et al.,1989, J. Biol. Chem. 264:20576-20582). In analogy to the soluble CR1system, soluble CR2 was produced in a recombinant system from anexpression vector containing the entire extracellular domain of thereceptor, but without the transmembrane and cytoplasmic domains. Thisrecombinant CR2 is reported to bind to C3dg in a 1:1 complex with Kdequal to 27.5 mM and to bind to the Epstein-Barr proteins gp350/220 in a1:1 complex with a Kd of 3.2 nM (Moore, et al., 1989, J. Biol. Chem.264:20576-20582).

CR3. A third complement receptor, CR3, also binds iC3b. Binding of iC3bto CR3 promotes the adherence of neutrophils to complement-activatingendothelial cells during inflammation (Marks, et al., 1989, Nature339:314). CR3 is also involved in phagocytosis, where particles coatedwith iC3b are engulfed by neutrophils or by macrophages (Wright, et al.,1982, J. Exp. Med. 156:1149; Wright, et al., 1983, J. Exp. Med.158:1338).

CR4. CR4(CD11) also appears to be involved in leukocyte adhesion(Kishimoto, et al., 1989, Adv. Immunol. 46:149-82).

DAF. DAF, or decay-accelerating factor, is a membrane protein thatappears to have similar action to C4Bp in bringing about afunctional-dissociation of C2b from C4b. DAF is linked to membranes viaa phosphatidyl inositol glycolipid, and its absence from red blood cellshas been shown to be a major causative factor in paroxysmal nocturnalhemoglobinuria. (Encyclopedia of Human Biology, Academic Press, Inc.1991). DAF binds to C3b/C4b as well as C3 convertases (EP 0512 733 A2).

DAF contains 4 SCRs followed by an O-linked glycosylation region, and isterminated with a glycolipid anchor (EP 0512 733 A2). Cells that expressDAF show substantial increases in resistance to complement-mediated celllysis (Lublin, D. M. et al., 1991, J. Exp. Med. 174:35; Oglesby, T. J.,et al., 1991; Trans. Assoc. Am. Phys. CIV:164-172; White, D. J. G., etal., 1992; Transplant Proc. 24:474-476).

MCP. MCP or membrane cofactor protein, like DAF, contains 4 SCRsfollowed by an O-linked glycosylation region. MCP is terminated witn anextra cytoplasmic segment (whose importance is unknown) a transmembraneregion and an intracellular domain (EP 0512 733 A2). Also, like DAF,cells expressing NCP confer substantial increases in resistance tocomplement-mediated cell lysis. (EP 0512 733 A2 and Lublin, D. M., etal., J. Exp Med (19) 174:35; Oglesby, T. J. et al., Trans Assoc Am Phys(1991) CIV:164-172; White, D. J. G., et al., Transplant Proc (1992)24:474-476).

FACTOR H. Factor H is a plasma protein that is exclusively orpredominantly composed of SCRs (Chung, L. P., et al., 1985, Biochem. J.230:133; Kristensen, T., et al., 1986, J. Immunol. 136:3407). Factor His a regulator of the alternative pathway. Factor H binds to C3b and tothe C3b portion of C3 convertases (C3b, Bb) (Encyclopedia of HumanBiology, supra) accelerating dissociation of Bb from these complexesthereby inactivating them. Factor H also regulates the use of C5 in theclassical pathway by competing with C5 for binding to C3b, thusinactivating the activity of the C3/C5 convertase (Encyclopedia of HumanBiology, supra).

2.3. SELECTINS AND SELECTIN LIGANDS

Selectins are a group of cell surface glycoproteins whichcharacteristically display a NH₂ terminal lectin domain related to thecarbohydrate recognition structure described for animal lectins, anepidermal growth factor domain, and a domain consisting of shortrepeating sequences analogous to those found in the complementregulatory proteins which map to a region of chromosome 1 called theregulators of complement activity (RCA) (Harlan & Liu, Adhesion: ItsRole in Inflammatory Disease, W. H. Freeman & Co., 1992). Threeindependently studied selecting have been characterized and are namedaccording to the cell type upon which each was originally identified.Under the current nomenclature there are the E-selectins, originallyidentified on cytokine-activated endothelial cells (Bevilacque, M. P. etal., (1985) J. Clin. Invest. 76:2003-2011); P-selectins, discovered onactivated platelets (Hsu-Lin, P. E., et al. (1984) J. Biol. Chem.259:9121-9126); and finally, L-selectins recognized as a cell surfacemarker on most leukocytes including lymphocytes, neutrophils, andmonocytes (Kansas, G. S. et al., (1985) J. Immunol. 134:2995-3002). Eachselectin has been implicated as a key factor in important events incellular adhesion and recognition. As such, their carbohydraterecognition structures at the NH₂-terminal portion of the molecule aswell as their carbohydrate ligands have been extensively studied.

Selectins, then, are cell adhesion molecules that in inflammatorysituations are responsible for the attachment of platelets andleukocytes to vascular surfaces and their subsequent infiltration intothe tissue. During a normal inflammatory response the leukocytes, inresponding to various signals, enter the tissue and phagocytize invadingorganisms. In various pathologic inflammatory diseases, such aspsoriasis and rheumatoid arthritis, this response may lead to seriousorgan tissue damage. Similarly, in reperfusion injury, invadingleukocytes are responsible for tissue damage. And, aside from theirinvolvement in inflammation, cell adhesion molecules on selecting play acentral role in other diseases such as tumor metastasis.

In inflammatory situations, all three selecting are implicated in therecruitment of leukocytes to the site of inflammation. Early events inthe inflammatory response include the recruitment of neutrophils to thesite of tissue damage. In normal situations, circulating lymphocytesbind to the vascular endothelium with low avidity. Under situations ofdistress however, as when the body has been invaded by a bacterialpathogen or when tissue damage has occurred, leukocytes interact withthe activated endothelium in another manner. First, up regulation ofselecting on endothelial cells and platelets occurs to control thelocalization of leukocytes to the inflamed endothelium. The initial stepof attachment of neutrophils to the endothelial cells lining the venulesis controlled by selecting and is known as neutrophil “rolling” (vonAndrian, U. H. et al., (1991) Proc. Natl. Acad. Sci., U.S.A.88:7538-7542; Smith, C. W., et al., (1991) J. Clin. Invest.,87:609-618). This “rolling” precedes the firm adhesion of leukocytes,especially neutrophils to the endothelium which is controlled by adifferent class of receptors known as the integrins. (Lawrence, M. B.and Springer, T. S. (1991) Cell 65: 859-873; von Andrain, U. H. et al.,(1991) Proc. Natl. Sci. U.S.A. 88:7538-7542; Larson R. S. and Springer,T. A. (1990) Immunol. Rev. 114:181-217). Extravasation of the cells intothe surrounding tissue proceeds after the aforementioned attachmentprocesses have each been accomplished.

One of the selecting, E-selectin (ELAM-1, endothelial cell adhesionmolecule, LECCAM-2) is expressed on endothelial cells followinginduction by cytokines such as interleukin-1β, tumor necrosis factor-α,lymphotoxin, bacterial endotoxins, interferon-γ and the neuropeptidesubstance-p (Harlan & Liu, supra). The expression of E-selectin onactivated endothelium requires de novo synthesis, peaks at 4-6 hours,and persists from 2-48 hours after initial stimulus. Activatedendothelia expressing the ELAM-1 receptor have been shown to bindneutrophils (Bevilacque M. P., et al. (1987) Proc. Natl. Acad. Sci.U.S.A. 84:9238-9242); monocytes (Walz, G. et al., (1990) Science 250:1132-1135): eosinophil (Kyan-Aung (1991) J. Immunol. 146:521-528): andNK cells (Goelz, S. E. (1990) Cell 63:1149-1356). Additionally,activated endothelium binds some carcinoma cells (Rice, G. E. andBevilacqua M. P. (1989) Science 246:1303-1306; Walz, G. et al., (1990)250 1132-1135) implicating a role for E-selectins in attachment of tumorcells to blood vessel walls.

P-selectin (CD62, granule membrane protein-140, GMP-140, plateletactivation dependent granule external membrane, Padgem, LECCAM-3) isexpressed on activated platelets as well as endothelial cells. TheP-selectin expression can be mobilized from intracellular stores inminutes after activation. P-selectins bind neutrophils and monocytes, aswell as carcinoma cells (Walz, G., et al., (1990) 250:1132-1135).

P-selectin, or CD62 expression does not require de novo synthesisbecause this selectin is stored in secretory granules, also calledWeibel-Palade bodies, in both platelets and endothelial cells. Thus,within minutes of activation of either cell type, for example bythrombin, histamine, or phorbol esters, CD62 is rapidly transported tothe surface of the cell where it can bind the ligand found onneutrophils, monocytes, and other cells, These ligand-bearing cells thenadhere to the platelet or endothelial cells expressing the CD62receptor.

Patel et al. have found that endothelial cells also express CD62 inresponse to low levels of hydrogen peroxide or other oxidizing agentsthrough the production of free radicals (Patel et al., 1991, J. CellBiol. 112:749-759). While endothelial cells normally reinternalize CD62within minutes of activation, induction by free radicals producesprolonged expression of the selectin. Because neutrophils releaseoxidizing agents and free radicals following activation, initialrecruitment of neutrophils by transiently expressed CD62 couldeffectively prolong the expression of CD62 through free radicalgeneration by neutrophils (Harlan & Liu, Adhesion, supra).

L-selectin, (lymphocyte homing receptor, LECCAM-1, Mel-14, Leu-8, TQ-1,Ly-22, LAM-1) is constitute expressed on the cell surface and is shedafter activation (Jung. T. M. et al., (1988) J. Immunol.,141:4110-4117).

Recent advancements in the field of adhesion molecules have led to theunderstanding of the role of protein-carbohydrate interactions. Inparticular, the ligands for selectins have been recently studied(Bevilaque, M. P. and Nelson, R. M. (1993) J. Clin. Invest. 91:379-387). Among the ligands identified are the Lewis X blood antigen(Le^(x)) and sialylated Lewis X antigen. The Lewis X antigens have beenknown for some time, and had been identified as the terminal structureson cell surface glycoproteins and glycolipids or neutrophils andpromyelocytic cell lines (Harlan & Liu, Adhesion, supra).

Lowe et al. demonstrated that transfection of a cDNA for the Lewis bloodgroup fucosyl transferase (Galβ1,3/4GlcNAca1,3 fucosyltransferase) intoChinese hamster ovary (CHO) cells resulted in the expression of theLe^(x) and SLe^(x) antigens and the simultaneous ability of thetransfected cells to adhere to E-selectins on TNF-α-activated humanumbilical vein endothelial cells (HUVECs) (Lowe et al., 1990, Cell63:475-484). Sialidase treatment of the cells abolished their ability toadhere to activated HUVECs, indicating that a sialylated structure wasrequired for adhesion. Additionally, it was observed that apre-myelocytic leukemia-60 (HL-60) cell clone which expressed SLe^(x)bound to HUVECS while another clone that did not express SLe^(x) did notbind to HUVECS.

Phillips et al. produced CHO glycosylation mutants, which, unlike thewild-type cells, expressed fucosyltransferase activities thatsynthesized both Le^(x) and SLe^(x) (LEC11) or Le^(x) only (LEC12) asterminal sugar structures on cell surface glycoproteins (Phillips etal., 1990 Science 250:1130-1132). Only LEC11 cells bound to E-selectinon activated HUVECs, and the adhesion was abolished by pretreatment ofthe LEC11 cells with sialidase, implicating SLe^(x) as the ligand.

The nucleic acid sequence of an α1,3-fucosyl transferase responsible foradding a fucosyl residue to an appropriate carbohydrate such as ELAM,through an α1,3 glycosidic linkage has been reported (InternationalPatent Publication No. WO91/16900). This report also describesrecombinant COS and CHO cells transformed with the transferase.

Other ligands that bind to selectins have also been disclosed. Theseligands structurally resemble the Lewis X antigens (International PatentPublication No. WO92/02527 and International Patent Publication No.WO91/19502).

2.4. DISEASES INVOLVING INAPPROPRIATE COMPLEMENT ACTIVITY

Diminished expression of CR1 on erythrocytes of patients with systemiclupus erythematosus (SLE) has been reported by investigators fromseveral geographic regions, including Japan (Miyakawa, et al., 1981,Lancet 2:493-497; Minota, et al., 1984, Arthr. Rheum. 27:1329-1335), theUnited States (Iida, et al., 1982, J. Exp. Med. 155:1427-1438; Wilson,et al., 1982, N. Engl. J. Med. 307:981-986) and Europe (Walport, et al.,1985, Clin. Exp. Immunol. 59:547; Jouvin, et al., 1986, Complement3:88-96; Holme, et al., 1986, Clin. Exp. Immunol. 63:41-48). CR1 numberhas also been found to correlate inversely with serum levels of immunecomplexes, with serum levels of C3d, and with the amounts oferythrocyte-bound C3dg, perhaps reflecting uptake ofcomplement-activating immune complexes and deposition on the erythrocyteas an “innocent bystander” (Ross, et al., 1985, J. Immunol.135:2005-2014; Holme, et al., 1986, Clin. Exp. Immunol. 63:41-48;Walport, et al., 1985, Clin. Exp. Immunol. 59:547).

Abnormalities of complement receptor expression in SLE are not limitedto erythrocyte CR1. Relative deficiencies of total cellular CR1 ofneutrophils and plasma membrane CR1 of B lymphocytes of the SLE patientshave been shown to occur (Wilson, et al., 1986, Arthr. Rheum.29:739747).

The relative loss of CR1 from erythrocytes has also been observed inpatients with Human Immunodeficiency Virus (HIV) infections (Tausk, F.A., et al., 1986, J. Clin. Invest. 78:977-982) and with lepromatousleprosy (Tausk, F. A., et al., 1985, J. Invest. Dermat. 85:58s-61s).

Complement activation has also been associated with disease statesinvolving inflammation. The intestinal inflammation of Crohn's diseaseis characterized by the lymphoid infiltration of mononuclear andpolymorphonuclear leukocytes. It was found recently (Ahrenstedt, et al.,1990, New Engl. J. Med. 322:1345-9) that the complement C4 concentrationin the jejunal fluid of Crohn's disease patients increased compared tonormal controls. Other disease states implicating the complement systemin inflammation include thermal injury (burns, frostbite) (Gelfand, etal., 1982, J. Clin. Invest. 70:1170; Demling, et al., 1989, Surgery106:52-9), hemodialysis (Deppisch, et al., 1990, Kidney Inst.37:696-706; Kojima, et al., 1989, Nippon Jenzo Gakkai Shi 31:91-7), andpost pump syndrome in cardiopulmonary bypass (Chenoweth, et al., 1981,Complement Inflamm. 3:152-165; Chenoweth, et al., 1986, Complement3:152-165; Salama, et al., 1988, N. Engl. J. Med. 318:408-14). Bothcomplement and leukocytes are reported to be implicated in thepathogenesis of adult respiratory distress syndrome (Zilow, et al.,1990, clin Exp. Immunol. 79:151-57; Langlois, et al., 1989, Heart Lung18:71-84). Activation of the complement system is suggested to beinvolved in the development of fatal complication in sepsis (Hack, etal., 1989, Am. J. Med. 86:20-26) and causes tissue injury in animalmodels of autoimmune diseases such as immune complex-induced vasculitis(Cochrane, 1984, Springer Seminar Immunopathol. 7:263),glomerulonephritis (Couser et al, 1985, Kidney Inst. 29:879), hemolyticanemia (Schreiber and Frank, 1972, J. Clin. Invest. 51:575), myastheniagravis (Lennon, et al., 1978, J. Exp. Med. 147:973; Biesecker and Gomez,1989, J. Immunol. 142:2654), type II collagen-induced arthritis (Watsonand Townes, 1985, J. Exp. Med. 162:1878), and experimental allergic andhyperacute xenograft rejection (Knechtle, et al., 1985, Heart Transplant4(5):541; Guttman, 1974, Transplantation 17:383; Adachi, et al., 1987,Trans. Proc. 19(1):1145). Complement activation during immunotherapywith recombinant IL-2 appears to cause the severe toxicity and sideeffects observed from IL-2 treatment (This, et al., 1990, J. Immunol.144:2419).

Complement may also play a role in diseases involving immune complexes.Immune complexes are found in many pathological states including but notlimited to autoimmune diseases such as rheumatoid arthritis or SLE,hematologic malignancies such as AIDS (Taylor, et al., 1983, ArthritisRheum. 26:736-44; Inada, et al., 1986, AIDS Research 2:235-247) anddisorders involving autoantibodies and/or complement activation (Ross,et al., 1985, J. Immunol. 135:2005-14).

Soluble CR1 has been successfully used to inhibit complement activationIn a number of animal models: Moat, B. P., et al., 1992, Amer. Review ofRespiratory disease 145:A845; Mulligan, M. S., et al., 1992, J. Immunol.148:1479-1485; Yeh, C. G. et. al., 1991, J. Immunol. 146 250-256;Weisman, et al., 1990, Science 249:146-51; Pruitt, et al., 1991,Transplantation 52(5):868-73; Pruitt and Bollinger, 1991, J. Surg. Res.50:350-55; Rabinovici, et al., 1992, J. Immunol. 149:1744-50; Mulligan,et al., 1992, J. Immunol. 148:1479-1485; Lindsay, et al., 1992, Annalsof Surg. 216:677.

Studies of Weisman et al (1990, Science 249:146-151) have demonstratedthat sCR1 can prevent 90% of the generation of C3a and C5a in humanserum activated by the yeast cell wall component zymosan. Weisman, etal. (1990, supra) also utilized sCRI in the rat to inhibit complementactivation and reduce the damage due to myocardial infarction. SolubleCR1 also appears to inhibit the complement dependent process of thereverse Arthus reaction (Yeh, et al., 1991, J. Immuno. 146:250-256), andhyperacute xenograft rejection (Pruitt, et al., 1991, Transplantation52:868-873). Recent data (Moat, et al., 1992, Amer. Rev. RespiratoryDisease 145:A845) indicate that sCR1 is of value in preventingcomplement activation in an experimental model of cardiopulmonary bypassin the pig, a situation where complement activation has beendemonstrated.

Citation or identification of any reference of Section 2 of thisapplication shall not be constructed as an admission that such referenceis available as prior art to the present invention.

3. SUMMARY OF THE INVENTION

According to the present invention, compositions are provided whichcomprise, in their broadest aspect, a complement moiety and acarbohydrate moiety. These compositions are useful in treating diseasesor disorders involving complement, as well as inhibiting a primary eventin the inflammatory response such as blocking interactions betweenintercellular adhesion molecules and their ligands. In preferredaspects, it is an advantage of the present invention that thecompositions comprise a ligand for intercellular adhesion molecules. Thecomplement moiety can be any one of a number of proteins which can bindto a complement component, or which are related to a complement receptortype 1 by virtue of containing an SCR motif. The carbohydrate moiety canbe any one of a number of carbohydrates that bind to or preventinteraction with an intercellular adhesion molecule. This constructfacilitates localization of the complement protein to the site ofinjury, and advantageously allows for, inter alia, lower dosagetreatment. It is a further advantage of the present invention that thesame composition can interrupt an initial event in the inflammatoryresponse. Therefore, the complement protein comprising a cellularadhesion molecule ligand is also useful in treating inflammationmediated by intercellular adhesion, as well as complement relateddiseases or disorders.

The carbohydrate moiety of the compositions of the invention is attachedto the complement moiety by means of an extracellular event such as achemical or enzymatic attachment, or can be the result of anintracellular processing event achieved by the expression of appropriateenzymes. In certain embodiments, the carbohydrate moiety willspecifically bind to intercellular adhesion molecules. In oneembodiment, the carbohydrate binds to a particular class of adhesionmolecules known as the selectins. Thus, in a preferred aspect, theinvention provides for a composition comprising at least one complementmoiety and at least one carbohydrate moiety, which compositionpreferentially binds to a particular selectin. Among the selecting areE-selectin, L-selectin or P-selectin. Particularly preferred embodimentscomprise at least one complement moiety and at least one carbohydratemoiety wherein said carbohydrate moiety comprises an N-linkedcarbohydrate, preferably of the complex type, and more preferablyfucosylated and sialylated. In the most preferred embodiments, thecarbohydrate is related to the Lewis X antigen, and especially thesialylated Lewis X antigen.

In one embodiment, the complement moiety is a protein that contains atleast one short consensus repeat and more preferably binds a componentof the complement cascade and/or inhibits an activity associated withcomplement. In a more preferred embodiment, the complement moietycomprises all or a portion of complement receptor type 1. Preferably thecomplement protein is soluble complement protein. In a most preferredembodiment, the complement moiety is soluble complement receptor type 1(sCR1), or a fragment or derivative thereof.

The present invention further provides pharmaceutical compositionscomprising at least one complement protein and at least one carbohydratemoiety in admixture with a suitable pharmaceutical carrier. In apreferred embodiment, the complement protein is soluble and particularlysCR1 or fragments or derivatives thereof. In these preferredembodiments, the carbohydrate is an N-linked carbohydrate, andpreferably fucosylated and more preferably fucosylated and sialylated.Of these the Lewis X (Le^(x)) antigen or sialyl Lewis X (sLe^(x))antigens are particularly preferred.

The present invention also provides methods for producing thecompositions described herein. In one preferred embodiment, theInvention provides for expressing the complement proteins in a cellwhich glycosylates the complement protein with a Le^(x) antigen, orpreferably a SLe^(x) antigen, and recovering the protein. In anotherembodiment, the invention provides for modifying a complement protein bychemically linking the carbohydrate moiety to the protein, wherein saidcarbohydrate moiety is preferably a selectin ligand.

In yet another embodiment, the invention provides for treating a subjectwith a disease involving undesirable or inappropriate complementactivity. Such treatment comprises administering to a subject in need oftreatment, a pharmaceutical composition in accordance with the presentinvention, in an amount and for a period of time suitable to regulatesaid undesirable complement activity. Preferably, the carbohydratemoiety in such pharmaceutical compositions are selectin ligands such asLe^(x), and more preferably the ligand is SLe^(x). Treatments with thecomplement protein comprising the selectin ligand include, but are notlimited to, diseases or disorders of inappropriate complementactivation, especially inflammatory disorders. Such disorders includebut are not limited to postischemic reperfusion conditions, infectiousdisease, sepsis, immune complex disorders and autoimmune disease.

The compositions of the invention can be used in homing the complementmoiety, preferably CR1, and more preferably sCR1, to adhesion moleculessuch as selectins on activated endothelium, allowing for, inter alia, alower dose as compared to the use of sCR1 alone or its presentglycoforms. The compositions can then persist at the site ofinflammation, and thereby prevent further activation. Early neutrophiladhesion events which depend on selectin/ligand interaction may also beblocked, Additionally, the in vivo half life of the sCR1 may beprolonged. In a specific embodiment, a CR1 moiety blocks the convertasesC3 and C5 in both the classical and alternative pathways, and thusprevents the release of C5a. Preventing the release of C5a furtherinhibits, inter alia, neutrophil activation and chemoattraction.

It is yet another advantage that the compositions presented herein mayhave reduced antigenicity. This may be particularly relevant in thecontext of the preferred embodiments as described herein, as thecarbohydrates relating to Lewis X antigen may be more “natural” in theirglycosylation patterning as compared to other carbohydrate structures,e.g. those obtained from non-human host cells and the like.

3.1. ABBREVIATIONS

CR1—Complement receptor one.

CR2—Complement receptor two.

CR3—Complement receptor three.

CR4—Complement receptor four.

DAF—Decay-accelerating factor.

ELAM—Endothelial cell adhesion molecule.

Le^(x)—Lewis X antigen.

LHR—Long Homologous Repeat.

MCP—Membrane cofactor protein.

sCR1—Soluble complement receptor one.

SLe^(x)—Sialyl Lewis X antigen.

SCR—Short consensus repeat.

CD15—Lewis X antigen

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A Shows a coomasie-stained SDS-PAGE gel; Lane 1 is molecularweight standards; Lane 2 is control HL-60 cell lysate; Lane 3 containssCR1[des-A] produced from DUKX.B11 cells; Lane 4 contains sCR1[des-A]recovered from LEC-11 cells.

FIG. 1B Shows a chemiluminescence Western blot of the same gel asdescribed in A probed with CSLEX1, an antibody that reacts with thesLe^(x) carbohydrate determinant. Lane 4 (which contains a soluble formof sCR1 containing LHR's BC and D (sCR1[des-A]sLex recovered from LEC-11cells) shows two distinct bads representing different glycosylationforms of the sCR1[des-A]sLex.

FIG. 1C Is the same gel described in Figure B, stripped and probed withaffinity purified rabbit polyclonal antibody to CR1. The blot shows thatthe two glycosylation forms of sCR1[des-A] from Gel B above are alsoreactive with the anti-complement receptor type 1 antibodies.

FIG. 2A is a Coomassie Blue stained polyacrylamide gel pattern. Thepredominant bands at approximately 187 kd in lanes 2, and 4-6 are thesCR1[des-A] protein, lane 2 obtained from DUKX-B11 cells, and lanes 4-6obtained from LEC-11 cells.

FIG. 2B is the same gel as FIG. 2A, Western blotted and probed with andanti-sCR1[des-A] polyclonal serum. As expected, all lanes containingsCR1-[des-A], whether derived from DUKX-B11cells or LEC-11 cells arepoitive for sCR1[des-A].

FIG. 2C is the same blot as FIG. 2B stripped and reprobed with anantibody specific for the sialy-diLewis x antigen (FH6) represented bythe shorthand notationNeUNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3GlcNAc. As expected,only lanes 4-6 containing sCR1[des-A]sLex obtained from LEC-11 cells arepositive for the appropriate carbohydrate structure.

FIG. 3 shows the results of a static adhesion binding assay. The blackbars represent the sCR1[des-A] material obtained from DUKX-B11 cells.The bars with horizontal lines represent sCR1[des-A]sLex materialobtained form LEC-11 cells. The sCR1[des-A]sLex material inhibitedbinding of U937 cells to activated aortic endothelial cells in aconcentration dependent manner.

FIG. 4 describes the protective effects of sCR1, sCR1[des-A], andsCR1[des-A]sLex from lung injury induced by CVF. A. is permeability, ameasure of radiolabelled protein leakage from the blood vessels of thelung. B. is the measurement of the reduction over control of hemorrhageas measured by a radiolabelled red blood cell leakage into the lung fromthe blood vessel. C. is a measure of the accumulation of neutrophils inthe lung as estimated by measurement of myeloperoxidase activity.

5. DETAILED DESCRIPTION

The present invention is directed to compositions comprising at leastone complement moiety and at least one carbohydrate moiety. Thecompositions of the invention interact on a cellular level with cellsexpressing appropriate receptors. In certain preferred embodiments, thecarbohydrate moiety of the compositions will bind to a selectin.

For the sake of clarity, the present invention is described in detail insections relating to the various components of the compositions, methodsof producing such compositions as well as pharmaceutical preparationsthereof, functional assays for measurement of activity of thecompositions, and methods of diagnosis, treatment and prophylaxis usingthe compositions.

5.1. COMPLEMENT PROTEINS

“Complement moiety” within the scope of this invention means any proteinthat contains all or a portion of any protein associated with thecomplement cascade, or a protein that contains at least a portion of ashort consensus repeat. Certain useful complement proteins are describedin detail in Sections 2.1 and 2.2 of the Background of the Invention,and preferably include but are not limited to complete proteins or anyfragment of: complement receptor type 1 (CR1), which is the receptor forcomplement components C3b and C4b; complement receptor type 2 (CR2),which is the receptor for C3d; complement receptor type 3 (CR3), thereceptor for iC3b; complement receptor type 4 (CR4), which is specificto iC3b; complement receptor type 5 (CR5), which is specific for the C3dportion of iC3b, C3dg, and C3d; the C5a receptor (C5a-R); and receptorsfor C3a and C4a. In a preferred aspect, the invention is meant toinclude those members of the family of complement regulatory proteinsthat contain the conserved short consensus repeat (SCR) motif. SCRmotifs are found in complement receptor type 1 and in several otherC3/C4-binding proteins, most notably CR2, factor H, C4-binding protein(C4-BP), membrane cofactor protein (MCP), and decay accelerating factor(DAF). The genes for factor H, C4-BP, CR2, and DAF map to a region onchromosome 1 which has been designated “regulators of complementactivation” (RCA) (Hourcade, D., et al., 1989, Advances in Immunol.,45:381-416). Particular analogues of these regulators of complementactivation are found in Atkinson, et al., EPO Publication No. 0 512 733A2, published on Nov. 11, 1992. Thus, in a preferred embodiment, thecomplement protein contains at least one SCR and is able to bind to acomponent of complement. Such complement proteins will, in oneembodiment, bind to C3b or C4b or a fragment of C3 or C4, such as thoseproteins described above.

CR1 has been extensively studied, and a structural motif of 60-70 aminoacids, termed the short consensus repeat (SCR) has been discovered. TheSCR motif is tandemly repeated 30 times in the F-allotype of CR1, andadditional repeat cycles occur in other allotypes. The consensussequence of the SCR includes 4 cysteines, a glycine and a tryptophanthat are invariant among all SCRs. Sixteen other positions areconserved, with the same amino acid or a conservative replacement beingfound in over half of the other 30 SCRs (Klickstein, et al., 1987, J.Exp. Med. 165:1095-1112; Klickstein et al, 1988, J. Exp. Med.,168:1699-1717; Hourcade et al., 1988, J. Exp. Med. 168:1255-1270). Thedimensions of each SCR are estimated to be approximately 2.5-3.0 nm×2nm×2 nm.

Tandem repeats of SCRs (the same invariant residues and similar spacingbetween cysteines) have been identified in 12 additional proteins of thecomplement systems (Ahearn et al., 1989, Adv. Immunol. 46:183-219).These proteins share a capacity for interacting with C3, C4, or C5, theset of homologous complement proteins that are subunits of thealternative and classical C3-C4 convertases and the membrane attackcomplex, respectively. Complement-related proteins containing SCRs mayhave activating functions (Clr, Cls, Factor B and C2), negativeregulatory roles (Factor H, C4-BP, DAF, MCP, and CR1), serve as cellularreceptors capable of eliciting functions of phagocytes and lymphocytes(CR1 and CR2) or promote the formation of the complement channel-formingmembrane attack complex (C6 and C7). Thus, the SCR is one of the mostcharacteristic structures of the complement system. The finding of SCR'sin non-complement proteins, such as in an interleukin-2 receptor αchain, β2-glycoprotein 1, and factor XIII does not necessarily indicatea complement-related function, although this possibility has not beenexcluded.

It is within the scope of the invention that the compositions compriseone or more of the aforementioned SCRs, in any combination suitable toobtain a desired result. As additional criteria, those forms of thecomplement protein or fragments thereof that are readily absorbed bytissues, that are protected from rapid metabolism and/or that providefor prolonged half life, are preferentially selected in producing thecompositions of the invention. One skilled in the art may also effectmodifications of the protein formulation, to effect absorption. Thesemodifications include, but are not limited to, use of a prodrug andchemical modification of the primary structure (Wearley, L. L., 1991,Crit. Rev. in Ther. Drug Carrier Systems, 8(4):333). In minimizingmetabolism of the complement protein and thereby increasing theeffective amount of protein, such modifications include but are notlimited to chemical modifications and covalent attachment to a polymer(Wearley, L. L., 1991, supra).

The compositions of the present invention may be part of a deliverysystem such as liposomes. Delivery systems involving liposomes arediscussed in International Patent Publication No. WO 91/02805 andInternational Patent Publication No. WO 91/19501, as well as U.S. Pat.No. 4,880,635 to Janoff et al. These publications and patents provideuseful descriptions of techniques for liposome drug delivery.

The genes for the complement related proteins are readily available, forinstance the nucleic acid sequences and/or genes encoding the complementproteins of the present invention are known as, for instance; DAF,International Patent Publication No. WO89/01041 published Feb. 9, 1989;MCP, Lublin M. D., et al., 1988, J. Exp. Med. 168:181-194; and, CR2,Weis, J. J., et al., 1988, J. Exp. Med. 168:1047-1066. The CR1 gene andits encoded protein are provided for in International Patent PublicationNo. WO89/09220 published Oct. 5, 1989 and entitled “The Human C3b/C4bReceptor (CR1)”. Once the gene and its encoded protein are available,any number of techniques known in the art can be used to modify the geneitself or its encoded proteins. The invention is meant to include suchcomplement protein-related fragments, derivatives, and analogues. Thecomplement protein-related fragments, derivatives, and analogues for usein the composition and formulations of the invention can be produced byvarious methods known in the art. The manipulations which result intheir production can occur at the gene or protein level, or by methodsof chemical synthesis. For example, a cloned complement gene can bemodified by any of numerous strategies known in the art (Maniatis, T.,1982, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). The complement protein genesequence can be cleaved at appropriate sites with restrictionendonuclease(s) followed by further enzymatic modification if desired,isolated, and ligated In vitro. In the production of the gene encoding aderivative, analogue, or peptide related to a complement protein, careshould be taken to ensure that the modified gene remains within the sametranslational reading frame as the native complement protein gene,uninterrupted by translational stop signals, in the gene region wherethe desired complement inhibitory-specific activity is encoded.

Additionally, the complement protein gene can be mutated In vitro or invivo, to create and/or destroy translation, initiation, and/ortermination sequences, or to create variations in coding regions and/orform new restriction endonuclease sites or destroy preexisting ones, tofacilitate further in vitro modification. Any technique for mutagenesisknown in the art can be used, including but not limited to, in vitrosite-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem.253:6551), use of TABX linkers (Pharmacia), and the like methods.

Manipulations of the complement protein sequence may also be made at theprotein level. Any of numerous chemical modifications may be carried outby known techniques, including but not limited to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄, acetylation, formylation, oxidation, reduction, and thelike.

In a particular embodiment in which the complement protein is CR1, forexample, specific modifications of the nucleotide sequence of CR1 can bemade by recombinant DNA procedures that result in sequences encoding aprotein having multiple LHR-B sequences. See, e.g., International PatentPublication No. WO91/05047, published Apr. 18, 1991. Such valencymodifications alter the extent of C3b binding which may be important fordisorders associated with such functions, such as immune or inflammatorydisorders. For example, full-length CR1 or fragments thereof and relatedmolecules which exhibit the desired activity can have therapeutic usesin the inhibition of complement by their ability to act as a factor Icofactor, promoting the irreversible inactivation of complementcomponents C3b or C4b (Fearon, D. T., 1979, Proc. Natl. Acad. Sci.U.S.A. 76:5867; Iida, K. and Nussenzweig, v., 1981, J Exp. Med.153:1138), and/or by the ability to inhibit the alternative or classicalC3 or C5 convertases.

In another embodiment, specific portions of the sequences of CR1 thatcontain specific, well defined combinations of LHRs or SCRs can also begenerated. The activities of these compounds can be predicted bychoosing those portions of the full-length CR1 molecules that contain aspecific activity. The resulting fragments should, but need not contain,at least one of the functions of the parent molecule. Such functionsinclude but are not limited to the binding of C3b and/or C4b, in free orin complex forms; the promotion of phagocytosis, complement regulation,immune stimulation; the ability to act as a factor I cofactor; promotingthe irreversible inactivation of complement components C3b or C4b,(Fearon, D. T., 1979, Proc. Natl. Acad. Sci. U.S.A. 76:5867; Iida, K.and Nussenweig, V., 1981, J. Exp. Med. 153:1138); effecting immunecomplex clearance and/or by the ability to inhibit the alternative orclassical C3 or C5 convertases. In a specific embodiment, the CR1includes LHR's B, C and D and does not include LHR A.

In addition, analogues and peptides related to complement proteins canbe chemically synthesized. For example, a peptide corresponding to aportion of complement protein which mediates the desired activity (e.g.,C3b and/or C4b binding, immune stimulation, complement regulation, etc.)can be synthesized by use of a peptide synthesizer.

In particular embodiments of the present invention, such complementproteins, including derivatives, analogues or fragments thereof, whetherproduced by recombinant DNA techniques or by chemical synthetic methods,include but are not limited to those containing, as a primary amino acidsequence, all or part of the amino acid sequence of the nativecomplement protein including altered sequences in which functionallyequivalent amino acid residues are substituted for residues within thesequence, resulting in a silent change. For example, one or more aminoacid residues within the sequence can be substituted by another aminoacid of a similar polarity which acts as a functional equivalent,resulting in a silent alteration. Nonconservative substitutions can alsoresult in functionally equivalent proteins.

In one embodiment, substitutes for an amino acid within the complementprotein sequence may be selected from other members of the class towhich the amino acid belongs. For example, the nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid.

In a particular embodiment, nucleic acid sequences encoding a fusionprotein, consisting of a molecule comprising a portion of a complementprotein sequence plus a non-complement protein sequence, can beproduced. See, e.g., International Patent Publication No. WO91/05047.For example, further modifications of complement proteins containingLHRs or SCRs include the generation of chimeric molecules containingportions of the LHR and/or SCR sequences attached to other moleculeswhose purpose is to affect solubility, pharmacology or clearance of theresultant chimeras. Such chimeras can be produced either at the genelevel as fusion proteins or at the protein level as chemically producedderivatives. Chimeric molecules comprising portions of immunoglobulinchains and complement protein can contain Fab or (Fab′)₂ molecules,produced by proteolytic cleavage or by the introduction of a stop codonafter the hinge region in the heavy chain to delete the F_(c) region ofa non-complement activating isotype in the immunoglobulin portion of thechimeric protein to provide F_(c) receptor-mediated clearance of thecomplement activating complexes. Other molecules that may be used toform chimeras include, but are not limited to, other SCR containingproteins, proteins such as serum albumin, heparin, or immunoglobulin,polymers such as polyethylene glycol or polyoxyethylated polyols, orproteins modified to reduce antigenicity by, for example, derivatizingwith polyethylene glycol. Suitable molecules are known in the art andare described, for example, in U.S. Pat. Nos. 4,745,180, 4,766,106 and4,847,325 and references cited therein. Additional molecules that may beused to form derivatives of the biological compounds or fragmentsthereof include protein A or protein G (International Patent PublicationNo. WO87/05631 published Sep. 24, 1987 and entitled “Method and meansfor producing a protein having the same IgG specificity as protein G”;Bjorck, et al., 1987, Mol. Immunol. 24:1113-1122; Guss, et al., 1986,EMBO J. 5:1567-1575; Nygren, et al., 1988, J. Molecular Recognition1:69-74). Constructs comprising a plurality of short consensus repeatshaving a complement binding site, said constructs attached to animmunoglobulin chain or a soluble, physiologically compatiblemacromolecular carrier, are also suitable as the complement moietytaught herein. Preparation of these constructs is disclosed inInternational Patent Publication No. WO91/16437, herein incorporated byreference.

Isolation and recovery of encoded proteins may be effected by techniquesknown in the art. The complement proteins may be isolated and purifiedby standard methods including chromatography (e.g., ion exchange,affinity, and sizing column chromatography, high performance liquidchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. If thecomplement protein is exported by a cell that is producing it, aparticularly efficacious method for purification of the protein is asfollows: the cell culture medium containing protein is subject to thesequential steps of a) cationic exchange chromatography, b) ammoniumsulfate precipitation, c) hydrophobic interaction chromatography, d)anionic exchange chromatography, e) further cationic exchangechromatography and f) size exclusion chromatography.

In a more preferred embodiment, the instant invention relates to solubleCR1 molecules. As used herein, the term soluble CR1 molecules meansportions of the CR1 protein, which, upon expression, are not located inthe cell surface as membrane proteins. As a particular example, CR1molecules which substantially lack the transmembrane region are solubleCR1 molecules. In a specific emmbodiment of the invention, an expressionvector can be constructed to encode a CR1 molecule which lacks thetransmembrane region (e.g., by deletion of the carboxyl-terminal to theAspartate encoded by the most C-terminal SCR), resulting in theproduction of a soluble CR1 fragment. In one embodiment, such a fragmentcan retain the ability to bind C3b and/or C4b, in free or in complexforms. In a particular embodiment, such a soluble CR1 protein may nolonger exhibit factor I cofactor activity.

Soluble constructs carrying some or all of the binding sites of CR1 arealso envisioned. Such constructs will in certain preferred embodiments,inhibit activation of complement and the complement dependent activationof cells. For example, in a specific embodiment, a soluble CR1 moleculecan be used which retains a desired functional activity, asdemonstrated, e.g., by the ability to inhibit classicalcomplement-mediated hemolysis, classical C5a production, classical C3aproduction, or neutrophil oxidative burst in vitro. In one embodiment,such a fragment can retain the ability to bind C3b and/or C4b, in freeor in complex form. The sCR1 molecule so produced can contain the LHR-A,LHR-B, LHR-C, LHR-D, SCR29, SCR30, up to and including the first alanineresidue of the transmembrane region. In a preferred aspect of theinvention, the soluble CR1 protein has the characteristics of theprotein expressed by a Chinese hamster ovary cell DUX B11 carryingplasmid pBSCR1/pTCSgpt as deposited with the ATCC and assigned accessionnumber CRL 10052.

In a further specific embodiment, a CR1 molecule can be produced thatlacks the LHR-A region of the CR1 molecule. To this end, an expressionvector can be constructed to encode a CR1 molecule which lacks thetransmembrane region and SCRs 1-7, resulting in the production of asoluble CR1 fragment that would be expected to preferentially inhibitthe alternative pathway. The expression vector so constructed would beexpected to contain sites for primarily C3b binding. Therefore, such aconstruct would be expected to preferentially inhibit the alternativecomplement pathway as assessed by the in vitro hemolytic assaysdescribed herein.

In yet another embodiment, an expression vector can be constructed tocontain only SCR's 1-18 of the complement receptor type 1. Such aconstruct would be expected to have the full function associated withcomplement receptor type 1 by virtue of containing sites for binding C3band C4b. Such a product would be expected to inhibit the classical andalternative pathways of complement as assessed by the in vitro assaysdescribed herein. In yet another embodiment the construct may containonly SCR's 15-18. Such a construct would be expected to bind C3bprimarily, and preferentially inhibit the alternative pathway ofcomplement.

These constructs, as well as the other constructs of the application canhave advantages due to differences in glycosylation. Such differencesare expected to affect such parameters as the in vivo half-life of themolecule. One skilled in the art will recognize the potential sites forN-linked glycosylation will vary with the products of such constructs.Differences in glycosylation can be assessed by the functional assaysdescribed herein for their ability to block the binding of the naturalligand for the particular cellular adhesion molecule.

The complement proteins of the invention can be assayed by techniquesknown in the art in order to demonstrate their complement-relatedactivity. Such assays include but are not limited to the following Invitro tests for the ability to interact with complement proteins, toinhibit complement activity, or to selectively inhibit the generation ofcomplement-derived peptides:

(i) measurement of inhibition of complement-mediated lysis of cells, forinstance, red blood cells (IH50 assay)(International Patent PublicationNo. WO92/10096)

(ii) measurement of ability to inhibit formation of complementactivation products such as, C5a and C5a des Arg and/or measurement ofability to inhibit formation of C3a or C3a des Arg, or measurement ofability to inhibit formation of C5b-9, or sC5b-9 (International PatentPublication No. WO92/10096)

(iii) measurement of ability to serve as a cofactor for factor Idegradation of, for instance, C3b or C4b (Makrides et al., (1992)267:24754-24761, Weisman, H. F., et al. (1990) Science, 244:146-151).

(iv) measurement of ability to bind to C3b or other C3 derived proteins,or binding of C4b or other C4b derived proteins (Makrides et al, supra,Weisman et al, supra)

(v) measurement of inhibition of alternative pathway mediated hemolysis(AH50 assay)(International Patent Publication No. WO92/10096)

Any complement protein or fragment, derivative or analog thereof, inparticular a CR1 protein, that has any one of the activities associatedwith complement receptors is within the scope of this invention as thecomplement moiety of the compositions provided herein.

Activities normally associated with complement receptor type 1 are welldocumented in the art. For example, for soluble CR1 proteins, suchactivities include the abilities in vitro to inhibit neutrophiloxidative burst, to inhibit complement-mediated hemolysis, to inhibitC3a and/or C5a production, to bind C3b and/or C4b, to exhibit factor Icofactor activity, and to inhibit C3 and/or C5 convertase activity. Arepresentative disclosure of activities and assays are described interalia in International Patent Publication No. PCT/US89/01358, publishedOct. 5, 1989 as WO89/09220, supra; and entitled Weissman, et al., 1990,Science 249:146-151; Fearon, D. T. and Wong, W. W., 1989, Ann. Rev.Immunol. 1:243; Fearon, D. T., 1979, Proc. Natl. Acad. Sci. U.S.A.76:5867; Iida, K. and Nussenzweig, V., 1981, J. Exp. Med. 153:1138;Klickstein et al., 1987, J. Exp. Med., 165:1095; Weiss, et al., 1988, J.Exp. Med., 167:1047-1066; Moore, et al., 1987, Proc. Natl. Acad. Sci.84:9194; Moore, et al, 1989, J. Biol. Chem. 264:205-76).

5.2. CARBOHYDRATE STRUCTURES COMPRISING SELECTIN LIGANDS

The carbohydrate moiety of the compositions of the present invention maybe selected from a variety of carbohydrate structures. In preferredembodiments, this moiety is responsible for binding the complementmoiety to particular cell adhesion molecules, such as a selectin.Section 2.3 of the Background of the Invention details several selectinsthat the carbohydrate moiety suitably binds to. Carbohydrate moietiesthat bind to intercellular adhesion molecules, including selecting, arewell known in the art. For instance, International Patent PublicationNo. WO91/19502 published Dec. 26, 1991 and entitled “IntercellularAdhesion Mediators”; International Patent Publication No. WO92/02527published Feb. 20, 1992 and entitled “New Carbohydrate-BasedAnti-Inflammatory Agents”; International Patent Publication No.WO92/19735 published Nov. 12, 1992 and entitled “GLYCAM-1(Spg 50), ASelectin Ligand”; International Patent Publication No. WO92/01718published Feb. 6, 1992 and entitled “Functionally ActiveSelectin-derived Peptides and Ligands for GMP-140”; International PatentPublication No. WO91/19501 published Dec. 26, 1991 and entitled“Intercellular Adhesion Mediators” all present disclosure ofcarbohydrate molecules useful in the present invention: the publishedpatent applications are herein incorporated by reference. The synthesisand processing of carbohydrates is also well known in the art (Hubbard,S. C. and Ivatt, R. J. (1981) Ann. Rev. Biochem. 50:555-83 and thereferences cited therein; Goochee, C. F., (1991) Biotechnology,9:1347-1355, and the references cited therein; Kobata, A. (1992) Eur. J.Biochem. 209, 483-501, and the references cited therein). Accordingly,the carbohydrate moiety of the instant invention can efficientlyinteract with cell adhesion molecules.

Particular ligands for selectins have also been described (Howard, D.R., et al., (1987) J. Biol. Chem. 262:16830-16837, Phillips, M. L., etal., (1990) Science 250:1130-1132, Walz, G. et al., (1990) Science,250:1132-1135, Stanley, P., and Atkinson, P., (1986) J. Biol. Chem.263:11374-11381; Butcher, E., (1991) Cell, 67:1033-1036). The Lewis Xand sialyl Lewis X oligosaccharides have been shown to be particularlyimportant in selectin binding. Recent studies have further characterizedthe ligand structures for selectins and note that modifications of theLewis X and sialyl Lewis X oligosaccharide may enhance the interactionsbetween the oligosaccharides and the selectins (Bevilacqua, M. P. andNelson, R. M. (1993) J. Clin. Invest. 91:379-387, Nelson, R. M., et al.,(1993) J. Clin. Invest. 91:1157-1166, Norgard, K. E. et al., (1993)Proc. Natl. Acad. Sci., U.S.A. 90:1068-1072; Imai, Y. et al., (1993)Nature 361:555-557).

The carbohydrate moiety of the present invention will now be describedwith reference to commonly used nomenclature for the description ofoligosaccharides. A review of carbohydrate chemistry which uses thisnomenclature is found in, Hubbard and Ivatt (1981) supra. Thisnomenclature includes, for instance, Man, which represents mannose;GlcNAc, which represents 2-N-acetyl glucosamine; Fuc, which representsfucose; Gal, which represents galactose; and Glc, which refers toglucose. In preferred embodiments, the carbohydrate moiety comprisessialic acid residues. Two preferred sialic acid residues are describedin shorthand notation by “NeuNAc”, for 5-N-acetylneuraminic acid, and“NeuNGc” for 5-glycolyl neuraminic acid. (J. Biol. Chem., 1982,257:3347; J. Biol. Chem., 1982, 257:3352).

This method of describing carbohydrates, as will be readily understoodby one skilled in the art, includes notations for the various glycosidicbonds relevant to naming carbohydrates. Therefore, in describing a bondlinking two or more monosaccharides to form an oligosaccharide, a βglycosidic bond between the C-1 of galactose and the C-4 of glucose iscommonly represented by Galβ1-4Glc. The notation β and α are meant torepresent the orientation of the bond with respect to the glycosidicring structure. For the D-sugars, for instance, the designation β meansthe hydroxyl attached to the C-1 is above the plane of the ring.Conversely, for the D-sugars, the designation a means the hydroxyl groupattached to the C-1 is below the plane of the ring. The carbohydratemoiety will be described with reference to this shorthand notation.

In its broadest aspects, carbonydrate structures useful in the presentinvention may be selected from a wide range of structures. Preferably,the carbohydrate will interact at some level with an adhesion molecule.For example, such moiety will bind to, or prevent the binding of anatural ligand to a cellular adhesion molecule, or even displace anendogenously occurring ligand. As is well understood in the art,interaction between a particular ligand and its receptor is generallydescribed by affinity constants. “Binding affinity” is generallymeasured by affinity constants for the equilibrium concentrations ofassociated and dissociated configurations of the ligand and itsreceptor. The present invention contemplates such an interaction betweena carbohydrate ligand and its endothelial cell adhesion moleculereceptor. In general, the binding of the carbohydrate moiety shouldoccur at an affinity of about k_(a)=10⁴M⁻¹ or greater to be useful forthe present invention, with greater than about 10⁸M⁻¹ being morepreferable, and most preferably between about 10⁸M⁻¹ and about 10⁴M⁻¹.

In a particular embodiment, the carbohydrate structure of the presentinvention is a ligand for the class of cell adhesion molecules known asselecting. Selectins have been shown to bind to a variety ofcarbohydrate structures which can broadly be classified into threegroups. The first group includes the N-linked and O-linkedcarbohydrates. N-linked and O-linked carbohydrates differ primarily intheir core structures. The N-linked carbohydrates all contain a commonManα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAcβ-R core structure. Of theN-linked carbohydrates, the most important for the present invention arethe complex N-linked carbohydrates. Such complex N-linked carbohydrateswill contain several antennary structures. Thus, the mono-, bi-, tri-,tetra-, and penta-antennary outer chains are important. Such outer-chainstructures provide for additional sites for the specific sugars andlinkages that comprise the carbohydrates of the present invention.N-linked glycosylation refers to the attachment of the carbohydratemoiety via GlcNAc to an asparagine residue in the peptide chain.Therefore, in the core structure described, R represents an asparagineresidue. The peptide sequences of the complement moiety,asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine,wherein X is any amino acid except proline are possible recognitionsites for enzymatic attachment of the N-linked carbohydrate moiety ofthe invention. O-linked carbohydrates, by contrast, are characterized bya common core structure, which is the GalNAc attached to the hydroxylgroup of a threonine or serine.

The N-linked glycans are formed by a series of complex steps occurringintracellularly by a series of enzymes with the addition of appropriatesugars. Alternatively, the enzymatic synthesis of the core structurescan be accomplished extracellularly by chemical and enzymatic steps toproduce the appropriate carbohydrates. These chemical and enzymaticsyntheses have been described in the art, for instance in InternationalPatent Publication No. WO91/19502 and the references described therein,which is incorporated herein by reference.

Specific glycosyltransferases are important for the final outer chainstructures of the complex carbohydrates. These glycosyltransferases arehighly specific for the appropriate monosaccharides. Of particularimportance to the invention are the enzymes involved in sialylation andfucosylation of the Galβ1-4GlcNAc group found in the N-linked andO-linked oligosaccharides. It will be understood by one skilled in theart that terminal glycosylation sequences differ. Among the variousstructures found in the outer chain moieties of the complexoligosaccharide chains are the carbohydrates moieties that are known tobind to particular selectins.

Particularly preferred within the context of the present invention arethe sialylated, fucosylated N-acetylglucosamines which have both asialic acid and a fucose residue in specific position and linkage.Therefore, the oligosaccharides related to the Lewis X (Le^(x))carbohydrate (Galβ1-4(Fucα1-3)GlcNAc) are especially useful. Structuresof the general formula I are particularity relevant:

Especially significant among this group are the sialylated Lewis Xcarbohydrate determinant (sLe^(x)) Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAc andclosely related structures, including as well, sLe^(a), a structuralisomer of sLe^(x), Neu5Acα2,3Galβ1,3(Fucα1,4)GlcNAc. Therefore, in aparticularly preferred embodiment the carbohydrate structure isrepresented by the general formula II:

where R represents the remaining carbohydrate structure and SArepresents a sialic acid. In a preferred embodiment, the sialic acid is5-N-acetylneuraminic acid. In another embodiment, the sialic acid is5-glycolyl neuraminic acid.

Additional examples of specific carbohydrate structures useful in thecompositions of the invention are disclosed in International PatentPublication No. WO92/02527 and can be expressed as follows:

NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAc

NeuNAcα2-6 Galβ1-4(Fucα1-3)GlcNAc

NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc

NeuNAcα2-6 Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc

NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc

NeuNAcα2-6 Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc

NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-6 Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3galβ1-4(Fucα1-3)Glc

NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3galβ1-4(Fucα1-3)Glc

NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-3Galβ1-3(Fucα1-4)GlcNAc

NeuNAcα2-6Galβ1-3(Fucα1-4)GlcNAc

NeuNAcα2-3Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAc

NeuNAcα2-6Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAc

NeuNAcα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc

NeuNAcα2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc

NeuNAcα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-3Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-6Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-3Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-6Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc

NeuNAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc

NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc

NeuNAcα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc

NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc

NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)Glc

NeuNAcα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)Glc

NeuNAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

NeuNAcα2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc

Methods of chemically and enzymatically synthesizing the carbohydratestructure are well known in the art and can be found in InternationalPatent Publication No. WO91/19502 which is incorporated herein byreference. Additionally, these structures may be obtained by the methodsdescribed infra.

As will be described in detail in subsequent sections, these structuresmay be provided on the complement moiety by a variety of mechanismsincluding but not limited to the transfection of the particularcomplement expressing cell with appropriate fucosyltransferase enzymes.Alternatively, the structures may be chemically synthesized usingappropriate fucosyltransferases and sialyltransferases and chemicallylinked to the complement moiety. Such transferases are generallyavailable as described below.

As noted earlier, specific modifications of the selectin ligand mayenhance the interaction between the carbohydrate determinant andparticular selecting. Nelson et al. studied the binding interaction of aseries of oligosaccharides based on the SLe^(x) and SLe^(a) (sLe^(a) maybe especially significant in tumor metastasis due to its significantexpression on certain cancer cells) structures (Nelson, et al., (1993)J. Clin. Invest. 91:1157-1166). Nelson suggests that both the sialicacid and the fucose in specific position and linkage enhance E-selectinrecognition (Nelson, supra). Both the SLe^(a) and SLe^(a) contain aterminal sialic acid (Neu5Ac) linked in an α2-3 linkage to the galactose(Gal), which is in turn linked to N-acetylglucosamine (GlcNAc). Bothstructures also contain a fucose coupled to the sub terminal GlcNAc.This characteristic structure is generally a part of largerglycoproteins. Accordingly, in certain preferred embodiments, thecarbohydrate is modified to contain at least one sialic acid inconformation with at least one fucose residue.

E-selectin may also bind to oligosaccharides related to SLe^(x) andSLe^(a) which lack the terminal sialic acid but instead have a sulfategroup (Yuen, C. T. et al., (1992) Biochemistry 31:9126-9131).Modification of this primary structure may have selective advantage ofhoming the carbohydrate moiety to a particular selectin. Therefore,within the scope of the present invention are the carbohydrates whichlack the terminal sialic acid but instead have a sulfate group.Additionally, sulphation of glycoproteins may enhance ligand binding toL-selectins (Imai, Y, Lasky, L. A., and Rosen, S. D. (1993) Science361:555-557). In this regard, Yuen at al., (1992) Biochemistry31:9126-91341 is instructive and is hereby incorporated by reference.

Selective oxidization of the sialic acid residues, without affecting theunderlying oligosaccharide, enhances the interaction with L-selectins asdescribed by Norgard et al., (1993) Proc. Natl. Acad. Sci. U.S.A90:1086-1072, which is incorporated herewith by reference. Othermodifications of the primary structure which may result in enhancedbinding or selective binding of the carbohydrate bearing complementprotein are also within the scope of this invention.

Carbohydrate moieties within the scope of the present invention may alsoinclude carbohydrates that by virtue of structural modifications areindicated to provide a stabilized carbohydrate moiety having a structuremore resistant to metabolic degradation than the corresponding naturallyoccurring carbohydrate moiety. Such modified structures may also exhibita high affinity for the particular targeted cell adhesion molecule.Thus, the carbohydrate moiety within the scope of the present inventionmay also encompass carbohydrates specifically designed to gain affinityfor particular intercellular adhesion molecules. Such carbohydrates canbe structurally modified carbohydrate or a mimetic of a carbohydratestructure such that the structural variant or mimetic has about the sameor better selectin binding activity, immunogenicity, and antigenicity asthe corresponding naturally occurring carbohydrate structure.Accordingly, any modification to a carbohydrate structure that enhancesinteractions dependent on carbohydrate structures for recognition andadhesion are within the scope of the present invention. Certaincarbohydrate and carbohydrate mimetics that are structural andfunctional variants of the naturally occurring carbohydrates are foundfor instance in International Publication No. WO 93/23031, publishedNov., 29, 1993, by Toyokuni, et al. Those skilled in the art ofcarbohydrate chemistry and carbohydrate mimetics will also recognizethose structures which are suitable within the context of the presentinvention based on the teachings herein.

The second group of carbohydrates that interact with selectins and thatare included in the present invention, are the phosphorylated mono andpolysaccharides such as mannose-6-phosphate. This phosphorylatedmonosaccharide, as well as the high molecular weight yeast derivedphosphomannon (PPME), appear to exclusively bind partners of theL-selectins, as P-selectins and E-selectins do not bind these molecules(Bevilacqua, M. P. and Nelson, R. N. (1993) J. Clin. Invest.91:379-387).

Finally, some sulfated polysaccharides such as heparin bind to selecting(Nelson, R. M. et al., (1993) J. Clin. Invest. 91:1157-1166).

The present invention contemplates at least one discrete carbohydrateunit attached to a portion of the complement moiety. One skilled in theart will recognize that a complement protein within the scope of theinvention may contain several sites of N-linked or O-linkedglycosylation for the attachment of sugar moieties. Therefore, theinvention is meant to include one or many carbohydrate units attached toany given complement moiety. Within a particular carbohydrate side chainof the carbohydrate moiety of the compositions, there will often beseveral sites for the particular primary structures to occur. Forinstance, the N-linked complex carbohydrates contain one or moreantennary structures that are possible locations for attachment of thespecific carbohydrate structures of the invention to the complementmoiety, and therefore, the amount of glycosylation of a particularcomplement moiety may vary greatly in accordance with the biologicalactivity one is attempting to achieve with the overall composition.

Differences in glycosylation patterns of the complement moieties areadvantageous in aiding one to assess a particular composition based onits in vivo activity. Accordingly, various factors such as half-life andabsorption may be assessed, and a particular composition chosen, basedon these properties. Conditions that affect glycosylation include butare not limited to such parameters as media formulation, cell density,oxygenation, pH, and the like. Alternatively, one may wish to amplify aparticular enzyme, such as those specific transferases involved inadding the carbohydrate residues in the appropriate position andlinkage.

Several methods known in the art for glycosylation analysis are usefulin the context of the present invention. Such methods provideinformation regarding the identity and the composition of theoligosaccharide attached to the peptide. Methods for carbohydrateanalysis useful in the present invention include but are not limited to:lectin chromatography; HPAEC-PAD, which uses high pH anion exchangechromatography to separate oligosaccharides based on charge; NMR,; massspectrometry; HPLC; GPC; monosaccharide compositional analyses;sequential enzymatic digestion. Additionally, three main methods can beused to release oligosaccharides from glycoproteins. These methodsare 1) enzymatic, which is commonly performed usingpeptide-N-glycosidase F/endo-β-galactosidase; 2) β-elimination usingharsh alkaline environment to release mainly O-linked structures; and 3)chemical methods using anhydrous hydrazine to release both N-andO-linked oligosaccharides.

Several methods presented here and known in the art are useful indetermining the affinity of the molecules for the particular selectin.Generally, a number of methods can be used to assay the ability of thecompositions of the inventions to inhibit intercellular adhesionmediated by selecting. The competition assays described in the ExampleSection, for instance, disclose specific methods. For instance, theability of the carbohydrate-bearing complement protein to inhibitadhesion of the natural cellular ligands to the cells expressing theparticular selectin can be used. Typically, the complement protein ofthe invention is incubated with the selectin bearing cells in thepresence of the natural ligand-bearing cells, wherein theselectin-bearing cells having been immobilized on a solid support.Inhibition of the cellular adhesion is then assessed by eithercalculating the amount of the bound complement moiety or assessing thedisplaced cells. In this regard, HL-60 cells and activated humanplatelets and endothelial cells are especially useful.

In a preferred embodiment, the complement moiety comprises all or aportion of the complement receptor type 1, and especially any solublefragment of complement receptor type 1 as described in Section 5.1infra. In a particularly preferred embodiment, the complement moietycomprises sCR1. This protein, in its full-length form, has 25 sites forN-linked glycosylation. In this embodiment, carbohydrate side chains areprovided on the sCR1 molecule, which chains comprise one or morecarbohydrate structures that can bind to or prevent the binding of aparticular ligand for an endothelial cell receptor. In particular, thesecarbohydrate moieties are ligand.s for selectins. In a particularlypreferred embodiment, these carbohydrate moieties are the Lewis Xoligosaccharides sialylated Lewis X oligosaccharides or a combination ofboth. One skilled in the art will understand that the amount ofglycosylation may be varied from complete saturation of the availableglycosylation sites to just a few of such sites.

5.3 PRODUCTION OF COMPLEMENT PROTEINS COMPRISING A SELECTIN LIGAND

The present invention provides various methods for production of thecompositions disclosed and claimed herein, methods for preparation ofcomplement protein having selectin binding activity, i.e. comprising aselectin ligand such as Le^(x), or more preferably SLe^(x).

5.3.1. COTRANSFECTION

As used herein, the term “cotransfection” refers to introduction of anucleic acid encoding at least one complement moiety and at least onenucleic acid encoding an enzyme capable of transferring fucose to alactosamine sequence. This results in co-expression of at least onecomplement moiety and the enzyme in the cells. Useful enzymes includethe α1,3 fucosyltransferases. These enzymes useful in adding theappropriate sugars in the appropriate linkage include, but are notlimited to α1,3 fucosyl transferase, α2,3 sialyl transferase, α2,6sialyl transferase, α2,6 sialyl transferase, β1,4 galactosyltransferase, β1,3 galactosyl transferase, and β1,4 N-acetyl glucosyltransferase. These may be readily obtained from Genzyme, Inc.,Cambridge, Mass., Sigma, St. Louis, Mo., the Albert Einstein College ofMedicine, New York, N.Y., Biogen, Inc., Cambridge, Mass., or the likesources. Genes for such transferases are continuously being cloned andmore are expected to be readily available in the future.

In a preferred method, a 1,3-fucosyl transferase has been foundparticularly useful for this purpose. The term “α1,3-fucosyltransferase” as used herein refers to any enzyme that is capable offorming the Le^(x) determinant, e.g., capable of transferring fucose tothe lactosamine sequence. In particular, the α1,3-fucosyl transferase ofthe invention can demonstrate any one of the known substratespecificities (see Harlan and Liu, Adhesion, supra). Preferably, thecell is a mammalian cell, such as COS or Chinese hamster ovary (CHO)cells.

Genes which express α1,3-fucosyl transferase can be obtained from avariety of sources (see Kukowska—Latallo et al., 1990, Genes Dev.4:1288-1303; International Patent Publication No. WO91/16900; andPaulson & Colley, 1989, J. Biol. Chem 264:17615-17618).

The nucleic acid coding for at least one complement protein and thenucleic acid coding for the α1,3-fucosyl transferase protein can beinserted into an appropriate expression vector, or in two vectors. Asused herein, the term “expression vector” refers to a vector whichcontains the necessary elements for the transcription and translation ofthe inserted protein-coding sequences. The necessary transcriptional andtranslational signals can be supplied by the native genes and/or theirflanking regions.

A variety of host-vector systems may be utilized to express theprotein-coding sequence, as long as the system provides forglycosylation of the complement moiety(ies) using the co-transfectedenzyme system. Potential host-vector systems include but are not limitedto mammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); or microorganisms such as yeast containing yeast vectors.The expression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.

In one embodiment, the expression vector or vectors contains areplication origin. In an alternative embodiment, the vector or vectors,which include at least one complement moiety and at least one enzyme,are expressed chromosomally, after integration of the complement proteinand the enzyme (e.g. the α1,3-fucosyl transferase) coding sequence intothe chromosome by recombination. One skilled in the art will understandthat it may be desirable to insert multiple genes encoding varioustransferase enzymes or other enzymes to ensure that at least one of theenzymes so inserted will be optimal for purposes described herein. Thus,the insertion of a multiplicity of genes encoding enzymes demonstratinga differential ability to glycosylate may be preferable to insertion ofonly one such gene. Also, one may desire to cotransfect more than onegene encoding a complement related protein to vary the constructs ofthis portion of the compositions of the invention.

Any method known in the art for the insertion of DNA fragments into avector may be used to construct an expression vector or vectorscontaining at least one gene for expression of a complement protein andat least one gene for expression of an appropriate enzyme, andappropriate transcriptional/translational control signals. These methodsmay include in vitro recombinant DNA and synthetic techniques and invivo recombinants (genetic recombination).

Expression of additional nucleic acid sequences encoding complementproteins or peptide fragments may be regulated by an additional nucleicacid sequence so that the complement proteins or peptides and the genefor the enzyme is expressed in a host transformed with the recombinantDNA molecule. For example, expression of a complement protein and anα1,3-fucosyl transferase may be controlled by any promoter/enhancerelement known in the art, but these regulatory elements must befunctional in the host selected for expression. Promoters which may beused to control gene expression include, but are not limited to, theSV40 early promoter region (Benoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42); plant expression vectorscomprising the nopaline synthetase promoter region (Herrera-Estrella etal., Nature 303:209-213) or the cauliflower mosaic virus 35S RNApromoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871), and thepromoter of the photosynthetic enzyme ribulose biphosphate carboxylase(Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elementsfrom yeast or other fungi such as the Gal 4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter, and the following animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz etal., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald,1987, Hepatology 7:425-515); insulin gene control region which is activein pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495),albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha1-antitrypsin gene control region which is active in the liver (Kelseyet al., 1987, Genes and Devel. 1:161-171), beta-globin gene controlregion which is active in myeloid cells (Mogram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic proteingene control region which is active in oligodendrocyte cells in thebrain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

In a preferred embodiment, at least one complement protein with at leastone gene for a transferase is expressed in mammalian cells and morepreferably in Chinese hamster ovary (CHO) cells (See, e.g. Stanley etal., 1990, J. Biol Chem. 265:1615-1622).

In a specific embodiment, genomic DNA from cells and plasmid DNA areprepared by standard methods (Maniatis) and dissolved in Tris-EDTA(10:1) buffer. If polybrene transfection is used, the cellular genomicDNA is sheared. The cells can be transfected by either the polybrene orthe calcium phosphate method (See, e.g. Stanley et al., 1990, supra).

The cotransfection can also be accomplished using the DEAE-dextranprocedure (see Lowe et al., 1990, Cell 63:475-484; Davis et al., BasicMethods in Molecular Biology, Elsevier Publishing Co., 1986).

An expression vector or vectors containing at least one complementmoiety and at least one nucleic acid insert for any appropriate enzyme,can be identified by four general approaches: (a) PCR amplification ofthe desired plasmid DNA or specific mRNA, (b) nucleic acidhybridization, (c) presence or absence of “marker” gene functions, and(d) expression of inserted sequences. In the first approach, the nucleicacids can be amplified by PCR with incorporation of radionucleotides orstained with ethidium bromide to provide for detection of the amplifiedproduct. In the second approach, the presence of a foreign gene insertedin an expression vector can be detected by nucleic acid hybridizationusing probes comprising sequences that are homologous to an insertedcomplement protein and α1,3-fucosyl transferase gene. In the thirdapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g.,β-galactosidase activity, thymidine kinase activity,resistance to antibiotics, transformation phenotype, occlusion bodyformation in baculovirus, etc.) caused by the insertion of foreign genesin the vector. In a specific example, if a complement protein or anα1,3-fucosyl transferase gene are inserted within the marker genesequence of the vector, recombinants containing the inserts can beidentified by the absence of the marker gene function. In the fourthapproach, recombinant expression vectors can be identified by assayingfor the activity of the gene product expressed by the recombinant. Suchassays can be based, for example, on the physical or functionalproperties of the gene products in vitro assay systems, e.g., complementinhibitory activity, or binding with antibody or a selectin (see Section5.1, supra, and Section 5.3 infra).

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Aspreviously explained, the expression vectors which can be used include,but are not limited to, the following vectors or their derivatives:human or animal viruses such as vaccinia virus or adenovirus; insectviruses such as baculovirus; yeast vectors; and plasmid and cosmid DNAvectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences in addition to adding thecarbohydrate moiety (e.g. a selectin ligand) or modifies and processesthe gene product in the specific fashion desired. Expression fromcertain promoters can be elevated in the presence of certain inducers;expression of the genetically engineered complement moiety and theenzyme product may be controlled. Furthermore, different host cells havecharacteristic and specific mechanisms for the translational andpost-translational processing and modification (e.g., glycosylation,cleavage [e.g., of signal sequence]) of proteins. Appropriate cell linesor host systems can be chosen to ensure the desired modification andprocessing of the foreign proteins expressed.

A vector or vectors containing at least one complement protein and atleast one nucleic acid sequence encoding an appropriate enzyme areintroduced into the desired host cells by methods known in the art,.e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, lipofection(lysosome fusion), use of a gene gun, or a DNA vector transporter (see,e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J.Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent ApplicationNo. 2,012,311, filed Mar. 15, 1990).

Both cDNA and genomic sequences can be cloned and expressed.

Once a recombinant which expresses the complement protein gene or geneswith the appropriate enzyme gene or genes is identified, the geneproducts should be analyzed. This can be achieved by assays based on thephysical, immunological, or functional properties of the product.

Recovery of the expressed protein product comprising the compositions ofthe invention may be achieved by standard methods of isolation andpurification, including chromatography (e.g., ion exchange, affinity,and sizing column chromatography, high pressure liquid chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins.

Any human cell can potentially serve as the nucleic acid source for themolecular cloning of the complement moiety gene or genes and the enzymegene or genes. Isolation of the genes involve the isolation of those DNAsequences which encode a protein displaying complement proteinassociated structure or properties, e.g., binding of C3b or C4b orimmune complexes, modulating phagocytosis, immune stimulation orproliferation, and regulation of complement. The DNA may be obtained bystandard procedures known in the art from cloned DNA (e.g., a DNA“library”), by chemical synthesis, by cDNA cloning, or by the cloning ofgenomic DNA, or fragments thereof, purified from the desired human cell(See, for example, Maniatis et al., 1982), Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.; Glover, D. M. (ed.), 1985, DNA Cloning: A Practical Approach, NRLPress, Ltd., Oxford, U.K. Vol. I, II). Cells which can serve as sourcesof nucleic acid for cDNA cloning of the genes include but are notlimited to monocytes/macrophages, granulocytes, B cells, T cells,splenic follicular dendritic cells, and glomerular podocytes. Clonesderived from genomic DNA may contain regulatory and intron DNA regionsin addition to coding regions; clones derived from cDNA will containonly exon sequences. Whatever the source, the genes should bemolecularly cloned into a suitable vector for propagation of the gene.

In a preferred embodiment, the C3b/C4b receptor (CR1) protein and theLewis X antigen are used. In a more preferred embodiment, the CR1protein and the sialyl Lewis X antigen is used. The CR1 gene and itsencoded protein are provided for in International Patent Publication No.WO89/09220 published Oct. 5, 1989 and entitled “The Human C3b/C4bReceptor (CR1)”. A suitable enzyme is α1,3-fucosyl transferase, whosegene and encoded protein are provided for in Lowe et al., 1992, J. Biol.Chem. 267:4152-4160. Other genes capable of expressing α1,3-fucosyltransferases are described in International Patent Publication No.WO91/16900 Kukowska- Latallo et al., 1990, Genes Dev. 4:1288-1303, andPaulson et al., 1989, J. Biol. Chem. 264:17615-17618.

In this preferred embodiment, selection of the cells co-transfected withα1,3-fucosyl transferase that are capable of glycosylating proteins withthe appropriate carbohydrate molecule can proceed by panning the cellswith the CD15 structure over platelets activated with thrombin.Platelets activated with for instance, thrombin, ADP, collagen, orepinephrine, express the selectin receptors CD62/PADGEM/GMP140. Boundcells are removed in the presence of a chelating agent such as EDTAsince the selectin/carbohydrate interaction is dependant on Ca++ andMg++. These released cells are then cloned and screened for theappropriate activity. In another embodiment, the cells underconsideration can be assayed for selectin binding activity in acompetitive assay for binding of HL60 or U937 cells to activatedplatelets.

Assays for directly screening for α1,3-fucosyl transferase activity canbe accomplished by a variety of means. For example, an assay can testthe ability of the α1,3-fucosyl transferase to link radioactivelylabelled fucose to an acceptor molecule (See International PatentPublication No. WO91/16900). Assays which test for α1,3-fucosyltransferase activity are also known in the art (see Stanley et al., J.Biol Chem., 1987, 262:16830-16837, Lowe et al., 1992, J. Biol Chem.267:4152-4160; Stanley et al., 1990, J. Biol Chem 265:1615-1622).

5.3.2. MUTAGENESIS

This invention also encompasses the use of chemical mutagenesis, by wellknown methods in the art, of an appropriate cell line that expresses acomplement protein to yield a cell line capable of producing acomposition in accordance with the present invention and preferably acomposition, comprising a complement protein and selectin ligand, suchas the Le^(x) antigen, and more preferably a SLe^(x) antigen. Onesuitable method envisioned in this invention is production of cell linesthat express a suitable enzyme, such as α1,3-fucosyl transferase, usingethyl methane sulfonate (Stanley et al., 198:3, Somatic Cell Genetics9:593-608).

Parental cell lines, such as CHO, which express the desired complementprotein can be mutagenized at 34° C. and 38.5° C. with ethyl methanesulfonate (EMS; Eastman Chemical Co., Rochester, N.Y.) at aconcentration of 100 μg/ml. Any cell line, preferably a mammalian cellline, which expresses the desired complement protein can be used,provided that mutagenesis can potentially induce one or more enzymes,such as the α1,3-fucosyl transferases, to glycosylate the complementprotein. For example, cells which endogenously express the complementprotein can be used, as well as transfected cells which are competent inexpressing the desired complement protein. See Example Section 6.4.1.for a specific embodiment.

Other methods of mutagenesis are well known in the art and can also beused (Maniatis, Ad, supra). Additionally, the cells may be subject toirradium and mutagenized cells selected in the techniques describedherein.

Methods for screening for mutagenized cells that express thecompositions of the invention are known in the art and are described inSection 5.2 and the sections following this one.

5.3.3. TRANSFECTION OF CELLS HAVING APPROPRIATE ENZYME ACTIVITY WITH ACOMPLEMENT PROTEIN

Cells expressing transferase enzymatic activity can be obtained frommany sources. For example, cells can be used which endogenously expressan appropriate enzyme such as α1,3-fucosyl transferase, or cells can betransfected with genes encoding such enzymes by the methods taught inSection 5.3.1 supra. Also, cells which have been previously mutagenizedto express enzymes necessary for glycosylation with a carbohydratemoiety, such as α1,3-fucosyl transferase can be used (See Section 5.3.2supra). or, previously transfected cells can also be used. Once cellsexpressing the appropriate enzyme are obtained they are transfected withthe complement protein gene by methods known in the art. In particular,complement proteins are described in Section 5.1, supra; methods ofintroducing nucleic acids encoding such proteins into a suitable hostcell that already expresses an appropriate enzyme activity are describedin Section 5.3.1. , supra.

In particular, it is envisioned that a nucleic acid encoding acomplement protein can be introduced into cell lines, which may thenexpress the compositions of the invention, and especially the HL-60(ATCC #CCL-240) and K562 (ATCC #CCL-243) cell lines.

5.3.4. CELL FUSION

Another method for obtaining the compositions of the invention is bycell fusion. The necessary competent cell lines which express anappropriate enzyme and a complement protein, i.e., as described inSection 5.3.1. through 5.3.3. supra, can be fused with each other usingstandard cell fusion techniques (see Current Protocols in MolecularBiology, Greene and Wiley-Interscience (1989)).

In a specific embodiment, cells that express a complement protein arefused with cells that have the enzymatic activity. A specific example ofthis embodiment is presented in the Example Sections below.

Preferably, when preparing the hybrid cells in accordance with such cellfusion techniques, one cell should be selected or engineered, e.g. viamutagenesis, to lack the hypoxanthine-guanine phosphoribosyl transferasegene. These cells will lack the activity to recycle purine via thesalvage pathway which utilizes PRPP. These cells should be provided inexcess so that fusion events will be unlikely to yield hybrid cell-lineswhich do not contain the mutant cells. Either cell line, e.g. the onewhich expresses α1,3-fucosyl transferase or the cell line whichexpresses complement protein, may be mutagenized. The cells which werenot mutagenized will maintain the ability to utilize the salvagepathway. Therefore, only the few hybrid cell-lines which do not containthe mutagenized cell line will survive in the HAT medium (due to thepresence of the aminopterin). By overwhelming the fusion with themutagenized cells, most of the non-mutagenized cells (“normal” cells)will fuse with the mutagens, only a few of the normal cells will nothave fused with the mutagens. All of the cells which result in a fusionof mutagen: mutagen will soon die off since these cells will have nomeans of utilizing the purine salvage pathway. Thus, this negativeselection will yield hybrid cell-lines which express α1,3-fucosyltransferase activity and complement protein.

5.3.5. IN VITRO MODIFICATIONS

The necessary competent cell lines which express an appropriatecomplement protein as described supra, can be used as a source of thecomplement protein for subsequent post-production modification,modification of the existing carbohydrate structures may be accomplishedusing any of the appropriate enzymes described supra, at, for instance,Section 5.3.1. In a particular embodiment post-production modificationoccurs in vitro under the appropriate conditions using GDP-fucose andthe appropriate α1,3 fucosyl transferase. Such transferases aredescribed supra. The modification described would be expected to yield afucosylated oligosaccharide on an existing core carbohydrate structuresuch as Galβ1-4 GlcNAc. The appropriate sialyl transferase along withthe appropriate sialic acids would be expected to add the terminalsialic acid residues to the appropriate core structures such asGalβ1-4GlcNAc or Galβ1-4(Fucα1-4) GlcNAc. The resulting carbohydratescan be analyzed by any method known in the art including those describedherein.

5.3.6. CHEMICAL MODIFICATION

The present invention further contemplates preparing the compositions ofthe invention by covalently coupling a carbohydrate moiety to thecomplement moiety using chemical synthesis techniques well known in theart.

Thus, complement protein of this invention can be glycosylated with thecarbohydrate ligand by chemical modification. This modification canresult in a glycoprotein in which the complement protein is directlylinked to the carbohydrate ligand or, in an alternative embodiment, aninert protein that has binding activity can be covalently cross-linkedto the complement protein, whereby the inert protein bridges to thecarbohydrate. If such an inert protein is used, it is preferably a shortconsensus repeat (SCR) since the SCR is a structural motif found on manycomplement proteins (see Section 5.1. supra), and therefore is likely tominimally affect the structure and function of the complement protein.

As can be appreciated by one of ordinary skill in the art, acarbohydrate moiety can be purified and collected from natural sources.An example of this process is disclosed for Le^(x) and sLe^(x) inStanley et al., J. Biol Chem. 263:11374 (1988), and see WO 91/19502(PCT/US91/04284) and WO 92/02527 (PCT/US91/05416). Purified complementprotein can also be obtained, as described in Section 5.1, supr.Alternatively, the carbohydrate moiety can be prepared synthetically(see Wong et al., 1992, J. Am. Chem. Soc. 114:9283, C. F. Borman, 1992,C & EN Dec. 7; p25).

The carbohydrate moiety from any source can be conjugated to thecomplement protein from any source, to obtain the compositions of thisinvention using chemical synthesis techniques. En particularly preferredembodiments, the carbohydrate moiety is Lewis X, and more preferably itis sialyl Lewis X. Preferably, the complement protein is CR1, and morepreferably, soluble CR1.

The chemical cross-linking of the selectin ligand to the complementprotein can proceed using a traditional cross-linking agent, such as,but not limited to molecules having one functional group that can reactmore than one time in succession, such as formaldehyde (althoughformaldehyde is not indicated for use due to its potentialcarcinogenicity), as well as molecules with more than one reactivegroup. As used herein, the term “reactive group” refers to a functionalgroup on the cross-linker that reacts with a functional group on thecomplement protein so as to form a covalent bond between thecross-linker and protein. The cross-linker should have a secondfunctional group for reacting with the carbohydrate moiety. The term“functional group” retains its standard meaning in organic chemistry.Preferably the cross-linking agent of the invention is a polyfunctionalmolecule, i.e., it includes more than one reactive group. Thepolyfunctional molecules that can be used are biocompatible linkers,i.e., they are non-carcinogenic, nontoxic, and substantiallynon-immunogenic in vivo. Polyfunctional cross-linkers such as thoseknown in the art and described herein can be readily tested in animalmodels to determine their biocompatibility.

The polyfunctional molecule is preferably bifunctional. As used herein,the term “bifunctional molecule” refers to a molecule with two reactivegroups. The bifunctional molecule may be heterobifunctional orhomobifunctional Preferably, the bifunctional molecule isheterobifunctional, allowing for vectorial conjugation of thecarbohydrate moiety and the complement moiety. Typically, thepolyfunctional molecule covalently bonds with an amino or a sulfhydrylgroup on the complement protein and a hydroxyl group, an amino analdehyde or a carboxylic acid on the carbohydrate moiety. However,polyfunctional molecules reactive with other functional groups on thecomplement protein, such as carboxylic acids or hydroxyl groups, arecontemplated in the present invention.

The homobifunctional molecules have at least two reactive functionalgroups, which are the same. The reactive functional groups on ahomobifunctional molecule include, for example, aldehyde groups andactive ester groups. Homobifunctional molecules having aldehyde groupsinclude, for example, glutaraldehyde (Poznansky et al., 1984, Science223:1304-1306) and subaraldehyde. Homobifunctional molecules having atleast two active ester units include esters of dicarboxylic acids andN-hydroxysuccinimide. Some examples of such N-succinimidyl estersinclude disuccinimidyl suberate and dithio-bis-(succinimidylpropionated), and their soluble bis-sulfonic acid and bis-sulfonatesalts such as their sodium and potassium salts. These chemicalshomobifunctional reagents are available from Pierce Chemicals, Rockford,Ill.

When a reactive group of a hetero-bifunctional molecule forms a covalentbond with an amino group, the covalent bond will usually be an amido ormore particularly an imido bond. The reactive group that forms acovalent bond with amino groups may, for example, be an activatedcarboxylate group, a halocarbonyl group, or an ester group. Thepreferred halocarbonyl group is a chlorocarbonyl group. The ester groupsare preferably reactive ester groups such as, for example, anN-hydroxy-succinimide ester group or that of N-maleimido-6-aminocaproylester of 1-hydroxy-2-nitrobenzene-4-sulfonic acid sodium salt(Mal-Sac-HNSA; Bachem Biosciences, Inc.; Philadelphia, Pa.).

Another functional group on the complement protein typically is either athiol group, a group capable of being converted into a thiol group, or agroup that forms a covalent bond with a thiol group. Free sulfhydrylgroups can be generated from the disulfide bonds of a complement protein(or peptide) that contains one or more disulfides. This is accomplishedby mild reduction of the protein molecule. Mild reduction conditions arepreferred so that the secondary and tertiary structure of the protein isnot significantly altered so as to interfere with the protein function.Excessive reduction could result in denaturation of the protein. Suchreactive groups include, but are not limited to, disulfides that canreact with a free thiol via disulfide transfer, e.g., pyridyl disulfide,p-mercuribenzoate groups and groups capable of Michael-type additionreactions (including, for example, maleimides and groups of the typedescribed in Mitra and Lawton, 1979, J. Amer. Chem. Soc. 101:3097-3110).The covalent bond will usually be a thioether bond or a disulfide. Thereactive group that forms a covalent bond with a thiol group may, forexample, be a double bond that reacts with thiol groups or an activateddisulfide. A reactive group containing a double bond capable of reactingwith a thiol group is the maleimido group, although others, such asacrylonitrile, are also possible. A reactive disulfide group may, forexample, be a 2-pyridyldithio group or a 5,5′-dithio-bis-(2-nitrobenzoicacid) group.

According to the present invention, for attachment to sulfhydryl groupsof reduced proteins, the substrate linkers can be modified by attachinga maleimide or disulfide group to one end of the linker. The unmodifiedsite on the linker is covalently attached to a functional group on thecarbohydrate moiety. For instance, the substrate linkers which are esteror amide linked to compounds as described (Partis et al., 1983, J. ProChem. 2:263; Means and Feeney, 1990 Bioconjugate Chem. 1:2-12).

Some examples of heterobifunctional reagents containing reactivedisulfide bonds include N-succinimidyl 3-(2-pyridyl-dithio)propionate(Carlsson, et al., 1978, Biochem J., 173:723-737), sodiumS-4-succinimidyloxycarbonyl-alpha-methylbenzylthiosulfate, and4-succinimidyloxycarbonyl-alpha-methyl-(2-pyridyldithio)toluene. Someexamples of heterobifunctional reagents comprising reactive groupshaving a double bond that reacts with a thiol group include succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate and succinimidylm-maleimidobenzoate.

Other heterobifunctional molecules include succinimidyl3-(maleimido)propionate, sulfosuccinimidyl4-(p-maleimido-phenyl)butyrate, sulfosuccinimidyl4-(N-maleimidomethyl-cyclohexane)-1-carboxylate, andmaleimidobenzoyl-N-hydroxy-succinimide ester. Many of theabove-mentioned heterobifunctional reagents and their sulfonate saltsare available from Pierce Chemicals, (supra). Additional informationregarding how to make and use these as well as other polyfuctionalreagents that may be obtained are well known in the art. For example,methods of cross-linking are reviewed by Means and Feeney, 1990,Bioconjugate Chem. 1:2-12.

The reactive groups of the cross-linking agent can be spaced via analkyl (including saturated and unsaturated) group, a cyclic alkyl group,a substituted alkyl or cyclic alkyl group, or an equivalent spacergroup, including a peptide sequence. In a specific embodiment, thecross-linking reactive groups are spaced from 0 to about 20 atoms fromeach other, although spaces of more than 20 atoms are also contemplated.

In another embodiment, carbohydrate side chains of complementglycoproteins may be selectively oxidized to generate aldehydes (see,e.g., Jackson, 1944, Organic Reactions 2:341; Bunton 1965, Oxidation inorganic Chemistry, Vol. 1 (Wiberg, ed.), Academic Press, New York, p.367; (Cooper, et al., 1959, J. Biol. Chem. 234:445-448). This ispreferred when the carbohydrate side chains are not selectin ligands.The resulting aldehydes may then be reacted with amine groups (e.g.,ammonia derivatives such as a primary amine, hydroxylamine, hydrazide,hydrazide, thiohydrazide, phenylhydrazine, semicarbazide orthiosemicarbazide) to form a Schiff base or reduced Schiff base (e.g.,imine, oxime, hydrazone, phenylhydrazone, semicarbazone orthiosemicarbazone, or reduced forms thereof).

Hydrazide cross-linking agents can be attached to a selectin ligand,e.g., Le^(x) or SLe^(x), via an ester or amide link or a carbon-carbonbond, and then reacted with an oxidized complement glycoprotein,containing an oxidized carbohydrate. This results in hydrazone formationand the covalent attachment of the compound to the carbohydrate sidechain of the glycoprotein via a cross-linker group.

Alternatively, a glycoprotein form of a complement protein can bereacted with an α1,3-fucosyl transferase in the presence of fucose toyield a fucosylated form of the complement inhibitory glycoprotein.

5.4. FUNCTIONAL ACTIVITY

The present invention further provides assays for evaluating thefunctional activity of the compositions of the invention. In particular,the invention provides certain useful functional assays for a CR1molecule comprising a selectin ligand such as Le^(x), or preferablySLe^(x). As used herein, the term “functional activity” refers toimmunological binding in addition to biological functions of a molecule.Physical-chemical assays are also envisioned for determining the natureof the complement moiety and the carbohydrate moiety of the compositionsof the invention.

In one embodiment, the activities of the complement moiety and thecarbohydrate moiety can be evaluated separately. Thus, a complementprotein comprising a carbohydrate moiety in accordance with theteachings herein may have similar or identical electrophoreticmigration, isoelectric focusing behavior, proteolytic digestion maps,C3b and/or C4b and/or immune complex binding activity, complementregulatory activity, effects on phagocytosis or immune stimulation, orantigenic properties as known for the complement inhibitory protein,e.g., as described in Section 5.1, supra. Similarly, the functionalactivity of the carbohydrate moiety can be assayed directly, e.g., asdescribed in Section 5.2, supra, and in International Patent PublicationNo. WO91/19502.

A number of currently available monoclonal antibodies can be usedaccording to the present invention to inhibit intercellular adhesionmediated by selecting. For instance, CSLEX-1 (see, Campbell et al., J.Biol. Chem. 259:11208-11214 (1984)), VIM-2, which recognizes a sequenceslightly different from SLe^(x) (see, Macher et al., supra), FH6(described in U.S. Pat. No. 4,904,596) (all references are incorporatedherein by reference) or SH₃ and SH₄ generated by Dr. S. Hakomori of theBiomembrane Institute in Seattle, Wash.

In another embodiment, the functional activities or physical-chemicalproperties of the complement moiety and the carbohydrate moiety areevaluated in the same assay. For example, in a specific embodiment, themolecular weight of a complement inhibitory protein comprising aselectin ligand can be estimated by PAGE, an increase in the apparentmolecular weight indicating attachment of the selectin ligand, such asLe^(x), or preferably SLe^(x), to the protein. In another embodiment, asandwich immunoassay can be used to assay the functional activity. Forexample, by using antibodies to a complement inhibitory protein and aselectin ligand, the composition may be identified. In a specificembodiment, infra, an antibody specific for CR1 is adsorbed to an assayplate. The putative soluble CR1 comprising the selectin ligand SLe^(x)or Le^(x) is added to the plate under conditions that allow antibodybinding. The presence of bound soluble CR1 comprising SLe^(x) or Le^(x)is detected by adding a CSLE^(x) antibody or anti-CD15 antibody,respectively, labelled with FITC, followed by an anti-FITC antibodylabelled with horseradish peroxidase. As will be readily understood byone of ordinary skill in the art, such sandwich immunoassay can beconfigured with the CLSE^(x) antibody or anti-CD15 antibody on the solidphase, or as a direct rather than an indirect assay. In yet a furtherembodiment, a Western Blot assay can be used to show that the productincludes a complement protein comprising a selectin ligand. In oneaspect, the apparent molecular weight of a protein detected in one lanewith an antibody to the complement protein and in another lane with anantibody to the selectin ligand can be compared. Results showingidentical molecular weight are indicative of a positive identificationof the molecule. In another aspect, the protein can be purified byaffinity chromatography, either on an anti-complement protein column oran anti-selectin ligand column, and the purified protein detected onWestern blotting with the alternative protein.

As can be readily appreciated by one of ordinary skill in the art, anyaffinity binding partner of a complement inhibitory protein or aselectin ligand of high enough affinity can be used in assays in placeof specific antibody molecules.

One skilled in the art will also understand that there may be other waysthe activity of the individual components of the compositions may beassayed, or the overall activity of the compositions as a whole may beassayed. These types of assays are informational with respect toachieving the desired overall functions of the compositions in a desiredsetting, such as the therapeutic arena. Accordingly, this Section, aswell as the Examples Section, is meant to be exemplary of certainwell-accepted techniques.

5.5. THERAPEUTIC COMPOSITIONS AND USES

One major advantage of the compositions of the present invention is thatthe carbohydrate moiety “homes” to inflamed endothelium, and thuslocalizes the composition to the site of tissue damage, therebypotentiating its anti-complement activity and also blockingneutrophil-endothelial cell interactions such as neutrophil rolling andextravasation. By providing for the homing of the complement protein tothe site of injury, resulting in its persistence there, the claimedcompositions advantageously allow for lower dosage treatment than wouldbe possible when dosing with either of the constituents alone. Thecompositions of the invention may also demonstrate an increased halflife in vivo and/or a great bioavailability.

Expression of selecting participates in the recruitment of cells tosites of inflammation. It is well-documented that multiple adhesionproteins and their ligands are required for the process of leukocyteadhesion to and extravasation across endothelial cells. For example,based on studies performed with known activators of the expression ofELAM-1 (inflammatory cytokines, endotoxin) and CD62 (thrombin,histamine, etc.), their expression is thought to represent inflammatoryand hemostatic responses to tissue injury.

Leukocyte traffic across the vessel walls to extravascular vasculartissue is necessary for host defense against microbial organisms orforeign antigens and repair of tissue damage. Under some circumstances,however, leukocyte-endothelial interactions may have deleteriousconsequences for the host. During the process of adherence andtransendothelial migration, leukocytes may release products such asoxidants, proteases, or cytokines that directly damage endothelium orcause endothelial damage by releasing a variety of inflammatorymediators (Harlan & Liu, supra). Some of these mediators, such as theoxidants, can directly activate complement which then feeds back tofurther activate the neutrophils through C3a and C5a. This leads tofurther tissue damage. Intervention of this process by a complementinhibitory protein “homed” into the endothelial microenvironment by itsselectin interaction, could help to stop or slow down this process.

Finally, sticking of single leukocytes within the capillary lumen oraggregation of leukocytes within larger vessels may lead tomicrovascular occlusion and may produce ischemia. Leukocyte-mediatedvascular and tissue injury has been implicated in the pathogenesis of awide variety of clinical disorders. Inhibition of leukocyte adherence toendothelium-“anti-adhesion” therapy-represents a novel approach to thetreatment of those inflammatory and immune disorders in which leukocytescontribute significantly to vascular and tissue injury. Studies in vitroindicate that close approximation of the leukocyte to the endothelialcell forms a protected microenvironment at the interface of theleukocyte and endothelial cell that is inaccessible to plasmainhibitors. Highly reactive oxidants, proteases, and phospholipaseproducts released by adherent leukocytes at the interface can react withand damage the endothelium. Inhibition of such firm adherence preventsformation of a protected microenvironment, and thereby reduces this typeof “innocent bystander” injury to endothelium. Inhibition of leukocyteadherence to endothelium will also prevent emigration to tissue, and,consequently, reduce tissue damage produced by emigrated leukocytes.Finally, inhibition of leukocyte adherence to endothelium or homotypicaggregation will prevent microvascular occlusion.

The pharmaceutical compositions of the present invention can be used toblock or inhibit cellular adhesion associated with a number ofdisorders. For instance, a number of inflammatory disorders areassociated with selectins expressed on vascular endothelial cells andplatelets. The term “inflammation” is used here to refer to reactions ofboth the specific and non-specific defense systems. A specific defensesystem reaction is a specific immune system reaction to an antigen.Example of specific defense system reactions include antibody responseto antigens, such as viruses, and delayed-type hypersensitivity. Anon-specific defense system reaction is an inflammatory responsemediated by leukocytes generally incapable of immunological memory. Suchcells include macrophages, eosinophils and neutrophils. Examples ofnon-specific reactions include the immediate swelling after a bee sting,and the collection of PMN leukocytes at sites of bacterial infection,e.g., pulmonary infiltrates in bacterial pneumonias and pus formation inabscesses).

Additionally, the pharmaceutical compositions of the present inventioncan be used to eliminate or block the complement injury occurring intransplanted organs. Organs prepared for transplant can be perfused withthe compositions of the present invention. Alternatively, organs fortransplantation may be stored in solutions containing the compositionsof the present invention. Such storage can occur during, for instance,transportation. In a further embodiment, the compositions may be used toflush the area from which transplant organs are removed, as from acadaver. Subsequent perfusion and/or storage are also envisioned.

Other treatable disorders include, e.g., rheumatoid arthritis,post-ischemic leukocyte-mediated tissue damage (reperfusion injury),frost-bit injury or shock, acute leukocyte-mediated lung injury (e.g.,adult respiratory distress syndrome), asthma, traumatic shock, septicshock, nephritis, vasculitis and acute and chronic inflammation,including atopic dermatitis, psoriasis, and inflammatory bowel disease.Various platelet-mediated pathologies such as atherosclerosis andclotting can also be treated. In addition, tumor metastasis can beinhibited or prevented by inhibiting the adhesion of circulating cancercells. Examples include carcinoma of the colon and melanoma. In theseembodiments, the complement moiety portion of the compositions actalmost as a carrier protein.

Compositions of the invention find particular use in treating thesecondary effects of septic shock or disseminated intravascularcoagulation (DIC). Leukocyte emigration into tissues during septic shockor DIC often results in pathological tissue destruction. Furthermore,these patients may have widespread microcirculatory thrombi and diffuseinflammation. The therapeutic compositions provided herein inhibitleukocyte emigration at these sites and mitigates tissue damage.

The inhibitors of selectin-ligand interaction, coupled withanti-complement action, also are useful in treating traumatic shock andacute tissue injury associated therewith. Because the selectins play arole in recruitment of leukocytes to the sites of injury, particularlyELAM-1 in cases of acute injury and inflammation, inhibitors thereof maybe administered locally or systemically to control tissue damageassociated with such injuries. Moreover, because of the specificity ofsuch inhibitors for sites of inflammation, e.g., where ELAM-1 receptorsare expressed, these compositions will be more effective and less likelyto cause complications when compared to traditional anti-inflammatoryagents.

The compositions of the invention can be administered to a subject inneed thereof to treat the subject by either prophylactically preventinga disease state or relieving it after it has begun. The pharmaceuticalcompositions of the invention may be administered in any suitablemanner, including parental, topical, oral, or local (such as aerosol ortransdermal) or any combination thereof. The compositions are preferablyadministered with a pharmaceutically acceptable carrier, the nature ofthe carrier differing with the mode of administration, for example, oraladministration, usually using a solid carrier and I.V. administration aliquid salt solution carrier.

The compositions of the present invention include pharmaceuticallyacceptable components that are compatible with the patient and theprotein and carbohydrate moieties of the compositions of the invention.These generally include suspensions, solutions and elixirs, and mostespecially biological buffers, such as phosphate buffered saline,saline, Dulbecco's Media, and the like. Aerosols may also be used, orcarriers such as starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders, disintegrating agents, and thelike (in the case of oral solid preparations, such as powders, capsules,and tablets).

As used herein, the term “pharmaceutically acceptable” preferably meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans.

The formulation of choice can be accomplished using a variety of theaforementioned buffers, or even excipients including, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin cellulose, magnesium carbonate, and the like.“Peglation” of the compositions may be achieved using techniques knownto the art (see for example International Patent Publication No.WO92/16555, U.S. Pat. No. 5,122,614 to Enzon, and International PatentPublication No. WO92/00748). Oral compositions may be taken in the formof solutions, suspensions, tablets, pills, capsules, sustained releaseformulations, or powders. Particularly useful is the administration ofthe compositions directly in transdermal formulations with permeationenhancers such as DMSO. Other topical formulations can be administeredto treat dermal inflammation.

A sufficient amount bf the compositions of the invention should beadministered to the patient to ensure that a substantial portion of theselectin ligand expected to cause or actually causing inflammation isregulated, as well as to ensure that an optimal concentration of thecomplement moiety is also delivered to the site, to combat inappropriatecomplement-related activity. In this way, inflammation can either beprevented or ameliorated. The selection of compositions, frequency ofadministration, and amount of composition so administered will be inaccordance with the particular disease being treated and its severity,the overall condition of the patient, and the judgement of the treatingphysician. Typical dosing regions will be analogous to treatment ofthese disease states by the use of antibodies and other biologicals.Typically, the compositions of the instant invention will contain fromabout 1% to about 95% of the active ingredient, preferably about 10% toabout 50%. Preferably, the dosing will be between about 1-10 mg/kg.About 1 mg to about 50 mg will be administered to a child, and betweenabout 25 mg and about 1000 mg will be administered to an adult. Othereffective dosages can be readily determined by one of the ordinary skillin the art through routine trials establishing dose response curves.

In determining the dosage of compositions to be administered, it must bekept in mind that one may not wish to completely block all of theselectin receptors, or may wish to completely block such receptors foronly a limited amount of time (i.e. only a few hours postischemicevent). In order for a normal healing process to proceed, at least someof the white blood cells or neutrophils must be brought into the tissuein the areas where the wound, infection or disease state is occurring.Thus, amount of the composition administered as a blocking agent must beadjusted carefully based on the particular needs of the patient whiletaking into consideration a variety of factors such as the type ofdisease that is being treated. For example, one may never desire thatthe neutrophils reoccur in an arthritic joint, but would expect suchreoccurrence at some point after a myocardial infarct, tissue crushinjury, and the like.

In a preferred embodiment, the present invention contemplatespharmaceutical compositions comprising a complement inhibitory proteincapable of binding to a selectin. Preferably, the pharmaceuticalcomposition comprises a soluble CR1 molecule comprising a selectinligand such as Le^(x), or most preferably SLe^(x). In one aspect, such asoluble CR1 molecule has LHRs A, B, C and D. In another aspect, such asoluble CR1 has LHRs B, C and D.

Accordingly, it is envisioned that the pharmaceutical compositions ofthe invention will be delivered to achieve elevation of plasma levels ofthe protein to treat diseases or disorders that involve inappropriatecomplement activity, whether or not inflammatory activity is alsoinvolved. Diseases or disorders involving complement that requiresystemic or circulating levels of complement regulatory proteins aredetailed in Section 2.2; supra and in Table I that follows.

TABLE I Systemic Diseases and Disorders Involving ComplementNeurological Disorders multiple sclerosis stroke Guillain Barre Syndrometraumatic brain injury Parkinson's Disease Disorders of Inappropriate orUndesirable complement Activation hemodialysis complications hyperacuteallograft rejection xenograft rejection interleukin-2 induced toxicityduring IL-2 therapy Inflammatory Disorders inflammation of autoimmunediseases Crohn's disease adult respiratory distress syndrome thermalinjury including burns or frostbite Post-Ischemic Reperfusion Conditionsmylyocardial infarction balloon angioplasty post-pump syndrome incardiopulmonary bypass or renal bypass hemodialysis renal ischemiamesenteric artery reperfusion after aortic reconstruction transplantorgan reperfusion Infectious Disease or Sepsis Organ Preservation ImmuneComplex Disorders and Autoimmune Diseases rheumatoid arthritis systemiclupus erythematosus (SLE) SLE nephritis proliferative nephritisglomerulonephritis hemolytic anemia myasthenia gravis

In particular, those disorders with may be treated by systematicadministration are described in section 2.2 supra. In specificembodiments, disorders associated with extended zones of tissuedestruction due to burn or myocardial infarct-induced trauma, and adultrespiratory distress syndrome (ARDS), also known as shock syndrome, canbe treated by parenteral administration of an effective amount of acomplement inhibitory protein comprising a selectin ligand in accordancewith the teachings herein.

Thus, an effective amount of a composition in accordance with thepresent invention is an amount effective to inhibit complement activity,in addition to its other effects.

In a preferred embodiment, the use of a complement inhibitory proteinwith selectin binding activity should be particularly helpful inanti-inflammatory therapy. Since the selectin ligand will home thecomplement inhibitory protein to the site of injury, it will preventneutrophil rolling. This is because the selectin ligand will bind toselectins on the blood vessel wall, thus preventing the adhesion ofleukocytes, particularly neutrophils.

For example, in a particularly preferred embodiment, by adding theSLe^(x) moiety, the sCR1 activity is localized to and the site of tissuedamage, thus potentiating its anti-C activity and also blockingneutrophil-endothelial cell interactions such as neutrophil rolling andextravasation.

In addition, the compositions can be used in the homing of CR1 to itsligand (the selectins) on activated endothelium, rendering lower dosesmore efficacious as compared to administration alone of sCR1 alone orand its present glycoforms. Heightened persistence of the SLe^(x)-sCR1at the site of inflammation is also achieved, thereby preventing furtheractivation. Early neutrophil adhesion events which depend onselectin/ligand interaction are also blocked. Finally, the in vivo halflife and/or bioavailability of the sCR1 is prolonged.

5.6. APPLICATION IN THE DIAGNOSTIC FIELD

The compositions of the present invention can be used to constitutedetection reagents capable of binding to released or shed, orcirculating complexes comprising a cellular adhesion molecule. Suchreleased, or shed or circulating adhesion molecules may be present aresult of activation of a particular cell comprising a cellular adhesionmolecule. It is well known that, for instance, L-selectins areconstitute expressed on the surface of cells and are rapidly shedfollowing activation (Bevilacqua, M. P., and Nelson, R. N. (1993)supra). Thus, selectins appear to be controlled by their appearance anddisappearance from the surface of cells. Circulating receptors that areshed upon activation may be assayed by techniques well known to thoseskilled in the art. An example of such assays is found in InternationalPatent Publication No. WO87/03600, published on Jun. 18, 1987 which isincorporated herein by reference. Such cellular adhesion molecules maybe physically distinct from the receptors present on the surface of thecell as, for instance, the product of an alternative splicing event thatresults in a receptor that lacks certain domains necessary forattachment to the cell membrane. Alternatively, such receptors may befragments or portions of the natural receptor, or may be associated withlarger membrane fragments. Further, such receptors may be present onintact cells.

The compositions of the present invention may be useful in detecting thepresence or absence of the receptors in the circulation, as in, forinstance, a serum sample or other sample from a patient suspected ofexpressing the receptor. Alternatively, the compositions may bedetectably labelled and used in in vitro or in vivo diagnostic imagingfor the presence of the cellular adhesion receptors.

In certain inflammatory conditions such as reperfusion injury, septicshock, and other chronic inflammatory diseases (such as for example,psoriasis and rheumatoid arthritis), the inflamed endotheliumparticipates in the recruitment of cells to the site of injury.Accordingly, the compositions and methods of the present invention areuseful in detecting the presence or absence of such inflammatoryconditions by virtue of their demonstrated ability to bind to theactivated cells and displace or prevent the binding of the naturalligand. In this embodiment, the composition of the present invention aredetectably labelled by techniques well known in the art.

In a further embodiment, the compositions of the present invention areimmobilized on a solid support and the presence or absence of certaincellular adhesion molecules is detected by measuring or calculating thdamount of binding that occurs. In this embodiment, certain monoclonalantibodies well known in the art may be used in conjunction with thecompositions.

The compositions can also be used to study inflammatory and complementmediated diseases or disorders by. virtue of their direct interactionwith mediators of inflammation as described herein. In particular, thecompositions can be used in either In vitro or in vivo methods. In invitro methods the samples may be fluid specimens or tissue specimens andcan include enzyme-linked assays, such as immunoperoxidase assays orstaining of tissue samples.

The compositions of the invention can be used as part of a kit,especially a diagnostic kit. Such a kit may include, for instance, thecompositions of the invention, as well as, components that aredetectably labelled, as for instance, monoclonal antibodies to theparticular cellular adhesion molecule. In one embodiment, the kitincludes one or more compositions, along with the appropriate dilutionand incubation buffers, a detectably labelled binding partner suitablefor use in a sandwich assay format, and a substrate reagent.

6. EXAMPLE 1

6.1. GENERATION OF A SOLUBLE DELETION MUTANT OF COMPLEMENT RECEPTOR 1

The following experiments detail the generation of several solubledeletion mutants of complement receptor type 1 useful in the presentinvention.

6.2 GENERATION OF A SOLUBLE DELETION MUTANT OF CR1 (SCR1[DES-A]) LACKINGLHR-A

Plasmid pBSABCD is described in International Patent Publication No.WO89/09220 “The Human C3b/C4b Receptor (CR1)” by Fearon, D. T., et al.,published Oct. 5, 1989; (see also, Klickstein, L. B., et al., (1988) J.Exp. Med 168:1699-1717). This plasmid harbors a full-length cDNA forhuman CR1 inserted as a 6.86-kilobase (kb) EcoRI-Eco-RV piece in theEcoRI-SmaI sites of pBluescript KS+ (Stratagene, La Jolla, Calif.);thus, the EcoRV and SmaI sites did not regenerate. pBSABCD was furthermodified by introducing a translational stop codon at the junction ofthe extracellular and transmembrane regions to yield pBL-sCR1 capable ofexpressing a soluble CR1 protein lacking the transmembrane andcytoplasmic domains. (International Patent Publication No. WO89/09220“The Human C3b/C4b Receptor (CR1)” by Fearon, D. T., et al., publishedOct. 5, 1989; Weisman, H. F., et al., (1990) Science 249:146-151).

pBL-SCR1 was digested with ClaI and BalI, and the resulting fragments(3.96 and 5.9 kb) were purified from low melting temperature agarosegel. Plasmid pBR322 was digested with ClaI and BalI, and the 2.9-kbfragment was purified from agarose gel and ligated to the 5.9-kbfragment from pBL-sCR1. The ligation mix was transformed into competentE. coli DH5α cells (GIBCO BRL), and the resulting plasmid, pBR8.8, waspurified and digested with XbaI, generating two fragments of 7.45 and1.35 kb. The 7.45-kb fragment was purified and religated into a circularform. The resulting plasmid, pBR7.45, was digested with ClaI and BalI,and the 4.5-kb fragment containing the CR1 cDNA was ligated to the3.96-kb fragment from pBL-sCR1 generating pBL-sACD lacking LHR-B.

Digestion of pBL-CR1c2, also referred to as pBL-sACD (Makrides et al.,(1992) J. Biol. Chem. 267:24754-24761) with NarI and NsiI removed 76 bpfrom the 3′ end of the leader, the entire LHR-A, and 57 bp from the 5′end of LHRC; the 7.07 kb fragment was purified from agarose gel andligated to two synthetic double-stranded oligonucleotides (OperonTechnologies, Alameda, Calif.), 68 and 66 bp in length having thefollowing sequence:

1. 5′-  CG CCC GGT CTC CCC TTC TGC TGC GGA GGA TCC3′-     GGG CCA GAG GGG AAG ACG ACG CCT CCT AGG       CTG CTG GCG GTT GTG GTG CTG CTT GCG GTG       GAC GAC CGC CAA CAC CAC GAC GAA CGC GAC        CCG GTG         -3′  [SEQ ID NO. 1]        GGC CAC CGG ACC -5′  [SEQ ID NO. 2] 2.5′-  GCC TGG GGT CAA TGT CAA GCC CCA GAT CAT3′-          CCA GTT ACA GTT CGG GGT CTA GTA    TTT CTG TTT GCC AAG TTG AAA ACC CAA ACC    AAA GAC AAA CGG TTC AAC TTT TGG GTT TGG     AAT GCA -3′  [SEQ ID NO.3]     TT      -5′  [SEQ ID NO. 4]

(Operon Technologies, Alameda, Calif.). These oligonucleotides restoredthe missing sequences from both the leader and LHR-C, respectively. Inaddition, a single nucleotide change was designed in one of theoligonucleotides, such that the first codon of LHR-C in SCR 15 coded forglutamine, instead of the native histidine. The rationale for thismodification was two-fold: (1) to ensure that the junction between theleader peptide and the coding region of the mature protein would be thesame as in the native sCR1 (i.e. Glycine/Glutamine) thus avoidingpotential difficulties with cleavage of the leader by signal peptidases;(2) to ensure that the N-terminal amino acid in the processed proteinwould be the same as in the native CR1 (Klickstein et al., (1988) J.Exp. Med. 168: 1699-1717) thus minimizing the potential forimmunogenicity. The ligation mix was transformed into Escherichia colistrain DH5α (Gibco BRL, Gaithersburg, Md.) to produce plasmid pBL-CR1c8containing the leader, LHR-C and LHR-D.

pBL-CR1c8 was linearized with NsiI and dephosphorylated using bacterialalkaline phosphatase (Gibco BRL) according to the manufacturer'sinstructions. pBL-CR1c, also referred to as pBL-sCR1 [Weisman et al.,(1990) Science 249:146-151] was digested with NsiI and the 1.35 kbfragment containing most of LHR-B and the first 56 nucleotides fromLHR-C was purified from agarose gel, and ligated to the linearizedpBL-CR1c8. This effected the assembly of pBL-CR1c6A containing LHRs B,C, and D. The correct orientation of the BCD insert was determined byrestriction digestion analysis.

The insert was excised by digestion with XhoI, and purified from agarosegel. The expression plasmid pTCSgpt (International Patent PublicationNo. WO89/09220 “The Human C3b/C4b Receptor (CR1)” by Fearon, D. T. etal., published Oct. 5, 1989; Carson et al., (1991) J. Biol. Chem. 266:7883-7887) was digested with XhoI, dephosphorylated using bacterialalkaline phosphatase, and ligated to the BCD fragment. The ligation mixwas transformed into E. coli DH1, generating plasmid pT-CR1c6A. Thecorrect insert orientation was determined by BglI restriction digestion,and pT-CR1c6A was prepared on large scale. pT-CR1c6A is a plasmid whichharbors the coding sequence for the soluble deletion mutant of CR1lacking the LHR-A as well as the transmembrane and cytoplasmic domains.The resulting soluble deletion mutant is termed sCR1[des-A] containingLHR's B, C, and D.

7. EXAMPLE 2

7.1 CONSTRUCTION OF A SOLUBLE DELETION MUTANT OF CR1 CONTAINING SCR'S15-18

A DNA fragment composed of the CR1 leader and Short Consensus Repeats(SCR) 15 through 18 was PCR-synthesized using pBL-CR1c8 as template[Makrides et al. (1992) J. Biol. Chem. 267, 24754-24761]. The 5′ “sense”primer hybridized to the pBluescript polylinker region upstream of theCR1 leader, and contained an XhoI restriction site, underlined:

5′-CCCCCCCTCGAGGTCGACGGTATCGATAAGC-3′ [SEQ ID NO. 5]

The 3′ “antisense” primer contained restriction enzyme recognitionsequences for BglII and NotI sites, underlined:

5′-TATCAAATGCGGCCGCTAAGAATACCCTAGATCTGGAGCAGCTTGGTAACTCTGGC-3′ [SEQ IDNO. 6]

The resulting 980-bp fragment was digested with XhoI and NotI, andligated into pBluescript KS(+) (Stratagene, La Jolla, Calif.) previouslyrestricted with XhoI and NotI. The ligation mix was transformed into E.coli DH5α competent cells (GibcoBRL, Gaithersburg, Md.) to yield plasmidpB-CR1(15-18) (3.86 kb). This was linearized at the 3′ terminus of SCR18 using BglII, and blunt-ended with mung bean nuclease (New EnglandBiolabs, Beverly, Mass.) used according to the manufacturer'srecommendations. The linearized plasmid was ligated to a syntheticdouble-stranded oligonucleotide (Operon Technologies, Inc., Alameda,Calif.) composed of the following two complementary strands:

5′-GATGAACTAGTCTCGAGAG-3′ [SEQ ID NO. 7]

5′-CTCTCGAGACTAGTTCATC-3′ [(SEQ ID NO. 8]

The double-stranded oligonucleotide restored the missing base-pairs fromthe 3′ terminus of SCR 18, and introduced a translational stop codon,followed by SpeI and XhoI restriction sites. The Ligation mix wastransformed into E. coli DH5α competent cells yielding plasmidpB-CR1(15-18A) (3.88 kb).

The DNA fragment composed of the CR1 leader and SCR 15-18 was excisedfrom pB-CR1(15-18A) by digestion with XhoI, purified from agarose gelusing the Geneclean Kit (BIO 101, La Jolla, Calif.), and ligated to theexpression vector pTCSgpt [Carson et al., (1991) J. Biol. Chem. 266,7883-7887] previously restricted with XhoI and dephosphorylated withcalf intestinal alkaline phosphatase (Boehringer Mannheim, Indianapolis,Ind.). Transformation of the ligation mix into E. coli DH1 competentcells yielded plasmid pT-CR1c12 (8.52 kb).

7.2 TRANSFECTION AND SELECTION OF STABLE CELL LINES

pT-CR1c12 was linearized by FspI digestion, phenol-extracted,ethanol-precipitated and resuspended in sterile water. 30 μg ofrecombinant plasmid was cotransfected with 3 μg pTCSdhfr [Carson et al.,(1991) J. Biol. Chem. 266, 7883-7887] into CHO DUKX-B11 cells deficientin dihydrofolate reductase [Urlaub and Chasin (1980) Proc. Natl. Acad.Sci. USA 77, 4216-4220] using electroporation with the Gene Pulser(Bio-Rad) at 960 μF and 230 V. The transfected cells were transferred tonon-selective α-Minimum Essential Medium (α-MEM) supplemented with 10%heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, 50μg/ml gentamicin, 4 mM glutamine (Gibco BRL), 10 μg/ml each ofthymidine, adenosine, and deoxyadenosine (Sigma, St. Louis, Mo.). Aftertwo days the cells were selected in α-MEM supplemented with 10% dialyzedfetal bovine serum, 1% penicillin-streptomycin, 50 μg/ml gentamicin, 4mM glutamine, 20 mM HEPES pH 7.0, 6 μg/ml mycophenolic acid, 250 μg/mlxanthine, and 15 μg/ml hypoxanthine (Sigma). Clones secreting SCR 15-18were identified by enzyme immunoassay (Cellfree® CD35; T Cell Sciences,Inc.), and the complement inhibitory activity of the proteins wasconfirmed using hemolytic assays [Yeh et al., (1991) J. Immunol. 146,250-256]. High-expressing clones were selected in growth mediacontaining methotrexate (Lederle, Pearl River, N.Y.).

8. EXAMPLE 3

8.1 CONSTRUCTION OF A SOLUBLE DELETION MUTANT OF CR1 LACKING LHR D(sCR1[des-D])

Plasmid pBSABCD was obtained from Dr. Douglas Fearon [Klickstein et al.,(1988) J. Exp. Med. 168, 1699-1717]. This plasmid harbors a full-lengthcDNA for human CR1 inserted as a 6.86 kilobase (kb) EcoR1-EcoRV piece inthe EcoR1-SmaI sites of pBluescript KS+ (Stratagene, La Jolla, Calif.);thus, the EcoRV and SmaI sites did not regenerate. pBSABCD was furthermodified as described [Weisman et al., (1990) Science 249, 146-151] toyield pBL-sCR1 capable of expressing a soluble CR1 protein lacking thetransmembrane and cytoplasmic domains.

An unique NruI restriction site was introduced in pBL-sCR1 at position4200 basepair (bp), i.e., at the junction of LHR-C and -D. The enzymesite was engineered by site-directed mutagenesis [Kunkel (1985) Proc.Natl. Acad. Sci. USA 82, 488-492] using the Muta-gene Phagemid Kit(Bio-Rad Laboratories, Melville, N.Y.). The 40-base phosphorylatedmutagenic oligonucleotide (New England Biolabs, Beverly, Mass.) had thefollowing sequence:

3′CGACACTTGAAAGACAAGCGCTACCAGTGACATTTTGGGG5′ [SEQ ID NO. 9]

The underlined bases are those which differ from the wild-type sequence.DNA templates were sequenced by the dideoxynucleotide chain terminationmethod [Sanger et al., (1977) Proc. Natl. Acad. Sci. USA 74, 5463-54673]using the Sequenase kit (U.S. Biochemical, Cleveland, Ohio).

The mutagenized plasmid pBL-sCR1N (9.8 kb) was digested with NruI andBglII, and the 7.8 kb fragment was isolated from agarose and ligated toa double-stranded synthetic oligonucleotide composed of the followingcomplementary strands:

5′-CGCTTAAGCTCGA-3′ [SEQ ID NO. 10]

5′-GATCTCGAGCTTAAGCG-3′ [SEQ ID NO. 11]

The double-stranded synthetic oligonucleotide restored the missing basepairs from the 3′ terminus of SCR 21 (LHR C), and introduced atranslational stop codon followed by XhoI and BglII restriction sites.The resulting plasmid pBL-CR1c7 (7.8 kb) was digested with XhoI, and theinsert was ligated into the expression vector pTCSgpt [Carson et al.,(1991) J. Biol. Chem. 266, 7883-7887] previously restricted with XhoIand dephosphorylated with bacterial alkaline phosphatase (GibcoBRL,Gaithersburg, Md.) used according to the manufacturer's recommendations.Transformation of the ligation mix into E. coli DH1 competent cellsyielded plasmid pT-CR1c7.

Transfection and Selection of Stable Cell Lines.

pT-CR1c7 was linearized by FspI digestion, phenol-extracted,ethanol-precipitated and resuspended in sterile water. 30 μg ofrecombinant plasmid was cotransfected with 3 μg pTCSdhfr [Carson et al.,(1991) J. Biol. Chem. 266, 7883-7887] into CHO DUKX-B11 cells deficientin dihydrofolate reductase [Urlaub and Chasin (1980) Proc. Natl. Acad.Sci. USA 77, 4216-4220] using electroporation with the Gene Pulser(Bio-Rad) at 960 μF and 230 V. The transfected cells were transferred tonon-selective α-Minimum Essential Medium (α-MEM) supplemented with 10%heat-inactivated fetal bovine serum, 1% penicillin-streptomycin, 50μg/ml gentamicin, 4 mM glutamine (Gibco BRL), 10 μg/ml each ofthymidine, adenosine, and deoxyadenosine (Sigma, St. Louis, Mo.). Aftertwo days the cells were selected in α-MEM supplemented with 10% dialyzedfetal bovine serum, 1% penicillin-streptomycin, 50 μg/ml gentamicin, 4mM glutamine, 20 mM HEPES pH 7.0, 6 μg/ml mycophenolic acid, 250 μg/mlxanthine, and 15 μg/ml hypoxanthine (Sigma). Clones secreting sCR1[desD]LHR's A, B, and C were identified by enzyme immunoassay (Cellfree® CD35;T Cell Sciences, Inc.), and the complement inhibitory activity of theproteins was confirmed using hemolytic assays [Yeh et al., (1991) J.Immunol. 146, 250-256]. High-expressing clones were selected in growthmedia containing methotrexate (Lederle, Pearl River, N.Y.).

9. EXAMPLE 4

9.1 GENERATION OF SOLUBLE CONSTRUCTS OF CR1 CONTAINING THE SLEXCARBOHYDRATE MOIETY

Any of the foregoing soluble deletion mutants of CR1 or other complementmoiety as defined herein can be manipulated to contain a carbohydratemoiety useful within the scope of the present invention. The followingexamples describe the generation of sCR1[des-A]sLe^(x) a solubledeletion mutant of CR1 lacking LHR-A and containing the sLexcarbohydrate moiety.

9.1.1. TRANSFECTION OF THE sCR1[des-A] CONSTRUCT INTO LEC-11 CELLS

Following linearization by FspI restriction digestion, pT-CR1c6A(Example 1, Section 6.2, Supra) was cotransfected into LEC11 cells(Campbell, C., and Stanley, P., 1984, J. Biol. Chem. 259:11208-11214)with FspI-linearized pTCSdhfr* containing an altered mouse dihydrofolatereductaise, cDNA that displays an abnormally low affinity formethotrexate (Simonsen, C. C. and Levinson, A. D. (1983) Proc. Natl.Acad. Sci. USA 80: 2495-2499). Clones secreting sCR1[desA]sLe^(x) wereidentified by enzyme immunoassay (Cellfree CD35; T Cell Diagnostics,Inc.), and the complement inhibitory activity of the protein wasconfirmed using hemolytic assays (Yeh et al., (1991) J. Immunol. 146:250-256). High-expressing clones were selected in growth mediacontaining methotrexate (Lederle, Pearl River, N.Y.).

9.1.2 Cell Culture Droduction of sCR1[des-A]sLe^(x).

CHO DUKX-B11 cells secreting sCR1[desLHR-A] or CHO LEC-11 cellssecreting sCR1[desLHR-A]sLe^(x) were grown in T-225 flasks in 1:1Dulbecco's modified Eagle's medium with high glucose/Ham's nutrientmixture F12 without hypoxanthine and thymidine (JRH Biosciences, Lenexa,Kans.) supplemented with 2.5% heat-inactivated fetal bovine serum(Hyclone, Logan, Utah). The pH of the media was adjusted to 7.8 to usingsodium bicarbonate to minimize sialidase activity present in theconditioned medium. The conditioned medium from these cultures washarvested three times a week by decanting, filtered, and frozen at −70°C. until purification. Productivity was monitored by ELISA.

9.1.3 Purification of sCR1[desA]sLe^(x).

Filtered cell culture supernatants containing sCR1[desA]sLe^(x) orSCR1[des-A] were buffer exchanged and concentrated by cross-floawultrafiltration (30,000 molecular weight cut-off), filtered again,applied to a S-Sepharosa Fast Flow cation exchange column, and elutedwith a high salt concentration (0.5 M sodium chloride). The cationexchange eluant was precipitated with ammonium sulfate, separated bycentrifugation, resuspended in PBS, and filtered. The filtrate wasadjusted to 0.8 M ammonium sulfate, loaded on a Butyl-Toyopearl 650Mcolumn and eluted with a step to 0.09 M ammonium sulfate. The eluant wasconcentrated using Centriprep-30 concentrators (Amicon), subjected tosize exclusion chromatography on a Toyopearl HW55F column, againconcentrated using Centriprep-30 concentrators, sterile filtered, andstored frozen at −70° C. The purification process was monitored byabsorbance at 280 nm and by ELISA. Protein purity was examined bySDS-PAGE with either Coomassie Blue or silver staining and scanningdensitometry. Endotoxin levels were determined using the LimulusAmebocyte Lysate assay (Associates of Cape Cod, Inc., Woods Hole,Mass.).

10. EXAMPLE 5

10.1 IN VITRO COMPLEMENT REGULATORY ACTIVITY OF SCR1[des-A]SLEX ANDSCR1[des-A]

The in vitro regulatory activities of sCR1[des-A]sLe^(x) were comparedto those of sCR1[des-A], which is the same protein except lackingsLe^(x) glycosylation and which has been shown to selectively inhibitalternative complement activation in vitro. sCR1[desA]sLe^(x) wasconstructed and expressed in LEC11 cells, and purified from cell culturesupernatants as described in the previous examples. sCR1[des-A] wasconstructed as described above and expressed in CHO DUKX-B11 cells asdescribed above for sCR1[des-A]sLex except that pT-CR1c6a wascotransfected into DUKX-B11 with FspI-linearized pTCSdhfr. sCR1[des-A]was purified from cell culture supernatants as described above forsCR1[des-A]sLex.

sCR1[desA]sLe^(x) and sCR1[des-A] competed equally for the binding ofdimeric C3b to erythrocyte CR1. sCR1[des-A]sLe^(x) and sCR1[des-A] wereequivalent in their capacity to serve as a cofactor in the factor Imediated degradation of the C3b α-chain. sCR1[des-A]sLe^(x) andsCR1[des-A] were equivalent in their capacity to inhibit alternativecomplement mediated erythrocyte lysis using C4-deficient guinea pigserum as a complement source. sCR1[des-A]sLe^(x) and sCR1[des-A] wereequivalent inhibitors of complement mediated erythrocyte lysis underconditions which allow classical pathway activation. Both, however, weresignificantly less effective inhibitors of classical pathway mediatedhemolysis than sCR1, a soluble recombinant protein containing the entireextracellular sequence of CR1. Thus, sCR1[desA]sLe^(x), likesCR1[des-A], is a selective inhibitor of alternative complement pathwayin vitro.

10.1.1 Complement Proteins and Antibodies.

Human C4, C3, C3b, and chemically cross-linked dimeric C3b (C3b2) wereprepared as described previously (Makrides et al., 1992, Scesney et al.,Eur. J. Immunol., 1996, 26:1729-1735). C4 was treated with methylamineto produce C4ma, a C4b-like form of the protein (Makrides et al., 1992;Law and Levine, 1980). C3b, C3b₂, and C4ma were radiolabeled with ¹²⁵Iusing Iodo-beads (Pierce Chemical Co.) according to the manufacturer'srecommendations. C4 deficient guinea pig serum was obtained commercially(Sigma).

10.1.2 C3b₂ Binding Studies.

The binding of sCR1[desA]sLe^(x) and sCR1[des-A] to ¹²⁵I-C3b₂ wasassessed by competition with native CR1 on human erythrocytes (Weismanet al., 1990; Makrides et al., 1992). Human erythrocytes were dilutedwith an equal volume of Alsever's solution (113 mM dextrose, 27 mMsodium citrate, 2.6 mM citric acid, 72 mM sodium chloride, pH 7) andstored at 4° C. until use. There was no difference in C3b₂ binding tofreshly drawn erythrocytes and those stored in Alsever's solution.Immediately prior to use, erythrocytes were washed three times with PBS,0.1% BSA, and 0.01% sodium azide. ¹²⁵I-C3b₂ (0.55 nM) was incubated witherythrocytes (4×10⁹ cells/ml) for 60 min on ice (0° C.) in the presenceof varying concentrations of sCR1, sCR1[des-A], sCR1[des-A]sLex C3b₂, orC3b. Bound and free ¹²⁵I-C3b₂ were separated by centrifugation throughdibutyl phthalate. Nonspecific binding was determined in the presence of0.71 mg/ml purified rabbit anti-sCR1 antibody.

RESULTS

C3b₂ Binding Studies.

The competition of sCR1[des-A], sCR1[des-A]sLex and sCR1 with ¹²⁵I-C3b₂binding to erythrocyte CR1 was assessed. From the concentration ofcompetitor required to inhibit maximal ¹²⁵I-C3b₂ binding by 50%,apparent dissociation constants (K_(d,app)) for sCR1, C3b₂, and C3b wereestimated to be 2×10⁻⁹ M, 3×10⁻⁸ M, and 6×10⁻⁷ M, respectively, valueswhich are similar to results obtained in earlier studies (Weisman etal., 1990; Wong and Farrel, 1991; Makrides et al., 1992).sCR1[des-A]sLe^(x) or sCR1[des-A] compete equally for ¹²⁵I-C3b₂ bindingto erythrocyte CR1.

10.1.3 Cofactor Activity for Proteolysis of Fluid Phase C3b or C4ma byFactor I.

The capacity of sCR1[desA]sLe^(x) or sCR1[des-A] to promote the specificproteolysis of the C3b or C4ma α-chain was assessed on SDS-PAGE (Wong etal., 1985; Weisman et al., 1990; Scesney et al., Eur. J. Immunol., 1996,26:1729-1735). ¹²⁵I-C3b (6.8×10⁻⁹ M) or ¹²⁵I-C4ma (5.6×10⁻⁸ M) wasincubated in PBS with factor I (0.25 μM) and varying concentrations ofeither sCR1[des-A]sLe^(x) or sCR1[des-A] for 20 min at 37° C. followedby 5 min on ice (0° C.). Under these conditions the proteolysis of theC4ma C3b α-chain was dependent on the concentration of cofactor. Theremaining intact C3b α-chain was separated on reduced SDS-PAGE and thebands were cut out and measured in a γ-counter.

RESULTS

Cofactor Activity for the Factor I Proteolysis of the C3b α-chain and ofthe C4ma α-chain.

The specific proteolysis of ¹²⁵I-C3b or ¹²⁵I-C4ma by factor I wasmonitored on SDS-PAGE under conditions in which the extent of α-chaincleavage was dependent on the concentration of cofactor, eithersCR1[des-A]sLe^(x) or sCR1[des-A]. The loss of the band representing theintact C3b α-chain required similar concentrations of eithersCR1[des-A]sLe^(x) or sCR1[des-A]. The loss of the band representing theintact C4ma α-chain also required similar concentrations of eithersCR1[des-A]sLe^(x) or sCR1[desLHR-A]. It can be concluded thatsCR1[des-A]sLe^(x) and sCR1[des-A] were equivalent in their capacity toserve as a cofactor in the Factor I mediated degradation of the C3bα-chain.

10.1.4 Hemolytic Assay for Inhibition of Classical and of AlternativeComplement Activation.

The inhibition of complement activation was assessed as previouslydescribed (Weisman et al., 1990; Yeh et al., 1991). Sheep erythrocytessensitized with rabbit anti-sheep erythrocyte antibodies (Diamedix,Miami, Fla.) were lysed using human serum as a complement source in 100mM HEPES, 150 mM sodium chloride, 0.1% BSA, pH 7.4. Sensitized sheeperythrocytes (10⁷ cells/ml), normal human serum (1 in 400 dilution), andvarying concentrations of sCR1[des-A] or sCR1[des-A]sLe^(x) wereincubated for 60 min at 37° C. in V-bottom microtiter plates, the cellspelleted by centrifugation, and the supernatants transferred to a flatbottom microtiter plate and the absorbance at 405 nm determined in orderto guantitate released hemoglobin. Samples were paired with identicalcontrols lacking human serum (complement-independent lysis). Bothsamples and controls were run in triplicate. Control values weresubtracted from sample values and the fractional inhibition wasdetermined relative to the uninhibited (no added sCR1[des-A]sLe^(x) orsCR1[des-A]) sample.

The inhibition of alternative pathway hemolysis was assessed using themodified method of Platts-Mills and Ishizaka (1974). Rabbit erythrocyteswere lysed using C4 deficient guinea pig serum as complement in 100 mMHEPES, 0.15 N sodium chloride, 0.1% bovine serum albumin, pH 7.4 withadded EGTA and Mg²⁺ to 8 mM and 5 mM, respectively. Rabbit erythrocytes(1.2×10⁷ cells/ml), C4 deficient guinea pig serum (1 in 8 dilution), andsCR1[des-A]sLe^(x) or sCR1[des-A] were incubated 60 min at 37° C. in aV-bottom microtiter plate, and released hemoglobin was determined asbefore.

Inhibition of Heinolysis by the Alternative Complement Pathway UsingC4-deficient Guinea Pig Serum.

To rule out interference from either pre-existing or newly generatedC4b, the alternative pathway lysis of rabbit erythrocytes was examinedusing C4-deficient guinea pig serum as a complement source. Equivalentconcentrations of sCR1, sCR1[des-A], or sCR1[desA]sLe^(x) were requiredto inhibit alternative complement-mediated erythrocyte lysis.

Inhibition of Hemolysis Initiated by the Classical Complement Pathway.

The inhibition of complement lysis of antibody-sensitized sheeperythrocytes required approximately equivalent concentrations ofsCR1[desA]sLe^(x) or sCR1[desLHR-A]. These concentrations, however, wereapproximately 50-fold higher than those required for inhibition by sCR1which contains LHR's A, B, C, and D.

11. EXAMPLE 6

11.1 ANALYSIS of sCR1[des-A]sLex

In this Example the the purified proteins sCR1[des-A] andsCR1[des-A]sLex are compared in Western blot analysis.

11.1.1 ANTIBODIES

sCR1 was prepared as previously described (Weisman et al., 1990; Yeh etal., 1991). Polyclonal rabbit anti-sCR1 antibodies were prepared andpurified as described (Makrides et al., 1992). CSLEX-1 (anti-sialylLewis^(x)) was obtained from Becton Dickinson. FH6 (anti-sialyldi-Le^(x)) was obtained from Dr. S. Hakomori (Biomembrane Institute,Seattle Wash.). DREG-56 (anti-E-selectin) was obtained from Endogen,Cambridge, Mass.; anti-CD15 (anti-Lex)was obtained from AMAC (Westbrook,Me.

11.1.2 Western Blot Analysis of sCR1[desA]sLe^(x).

Western Blot analysis was conducted acccording to the followingprocedure:

a) glycoproteins obtained from the transfection of LEC-11 cells with thesCR1[des-A] construct described above were run subjected to SDSpolyacrylamide gel electrophoresis under reducing and non-reducingconditions with appropriate controls and standards.

b) the glycoprotein bands were transferred to solid support membranes(Immobilon™) via semi-dry electrophoretic transfer (IntegratedSeparation Systems)

c) the membranes containing the transferred glycoproteins were blockedwith 1% non-fat dry milk proteins for 2 hrs., (or overnight) or otherblocking reagents such as bovine serum albumin (at about 2.0%) andgelatin (at about 0.3%). The latter blocking reagents are preferable toavoid complications due to potential SLe^(x) glycosylated proteinspresent in the milk solution (such as IgA).

d) the replicate membranes were then reacted with antibodies to CR1and/or sCR1[des-A], antibodies to Lex(DAKO, AMAC, CD15), antibodies toSLe^(x)(CSLEX-1, available from ATCC, and from Becton Dickinson), andisotype matched control antibodies for 2hrs.

e) the membranes were washed in wash buffer(PBS-tween) 4 times for about10-15 minutes each.

f) the membranes were then reacted with HRP-labelled anti-murineantibodies or anti-rabbit antibodies (all available from various vendorssuch as Bio-rad, Southern Biotech, Tago) for 2 hrs.

g) the membranes were washed as in (e).

h) the membranes are developed with HRP subtrate (Bio-rad, Sigma) tovisualize the glycoprotein bands reactive with each primary antibody,or, a chemilumincescent method referred to as “ECL” (EnhancedChemiluminescence, Amersham).

RESULTS

11.2 Western Blot Analysis.

The above-described technique using the ECL Western Blot procedure fromAmersham and antibodies to SLe^(x) (CSLEX1) and antibodies to CR1(rabbit polyclonal antibodies) was performed using the material derivedfrom LEC-11 cells transfected with the complement moiety termedsCR1[des-A] obtained through the method described above to yieldsCR1[desA] sLe^(x). The results of this Western Blot analysis clearlydemonstrated that sCR1[desA]sLe^(x) derived from LEC-11 cells (PamelaStanley, Albert Einstein College of Medicine) bears sLe^(x) moieties asdetermined by staining with CSLEX1 antibodies, (ATCC HB 8580, see U.S.Pat. No. 4,752,569) while material derived from transfection of thesCR1[des-A] construct into DUKX.B11 CHO cells does not.

FIG. 1B shows the results of this analysis, the first lane with materialin it contains molecular weight standards. The second lane containslysate derived from HL-60 cells (positive control for CSLEX1 mAb). Thethird lane contains sCR1[des-A] material derived from DUKX.B11 CHO cellsand the fourth lane contains sCR1[des-A]sLe^(x) material derived fromLEC-11 cells. Of the two lanes containing the sCR1[des-A] material, onlythat lane derived from LEC-11 cells (Lane 4) was identified by theCSLEX1 mAb as demonstrated by two clear bands consistent with twoglycosylation forms of sCR1[des-A]. Both lanes containing sCR1[des-A](Lane 3 from DUX.B11, and Lane 4 from LEC-11) reacted with a polyclonalantibody to sCR1[des-A] as expected (FIG. 1C). FIG. 1A is acoomasie-blue stained SDS-PAGE gel in the same material.

11.3 SECOND WESTERN BLOT ANALYSIS

In a separate experiment sCR1[desLHR-A] and sCR1[desA]sLe^(x) weresubjected to SDS-PAGE using a 4-20% gel (ISS) and non-reducingconditions. The gels were blotted onto a membrane (Immobilon-P) using asemi-dry transblotting apparatus (ISS). The membranes were blockedovernight at room temperature in a solution of tris buffered saline(TBS) containing 2% BSA, 1% normal goat serum, 0.05% sodium azide. Theblot was probed with FH6 (anti-sialyl di-Le^(x), Hakomori supernatant,diluted 1:1 in TBS blocking buffer) for 2 h at room temperature. Afterextensive washing in PBS with 0.05% Tween-20, the blot was probed withhorseradish peroxidase (HRP) conjugated goat anti-mouse IgM (Tago, 1ug/ml in blocking buffer) for 1 h at room temperature. After extensivewashing in PBS with 0.05% Tween-20, the blot was incubated with achemi-luminescent substrate (ECL kit, Amersham) for 1 minute, exposed tox-ray film for 30-120 s, and the film developed. The blot was thenstripped and re-probed with rabbit polyclonal anti-sCR1 (1:2000 inblocking buffer) for 1 h, washed extensively, probed with HRP conjugatedanti-rabbit Ig (Amersham), washed and detected as before.

RESULTS OF THE SECOND WESTERN BLOT ANALYSIS

In the second western blot analysis both CSLEX-1, a monoclonal antibodythat reacts with sLe^(x) oligosaccharides, and FH60, a monoclonalantibody that reacts with sialyl di-Le^(x), bound to sCR1[desA]sLe^(x)but not to sCR1[desA]. Both oligosaccharide structures have been shownto be ligands for selectins (Goelz, S. et al., J. Biol. Chem. (1994)269:1033-1040). Parekh et al. (1992) J. Biochem. (Tokyo) 16d,137,identified the carbohydrate strucures responsible for binding to anE-selectin affinity column to be sialyl di-Le^(x). No insight into thenumber of N-linked glycosylation sites used, or how many of thoseterminate in sLe^(x), can be derived from this experiment.

FIGS. 2A through 2C detail the results of the second Western blotexperiment. FIGS. 2A through 2C are an analysis of the samepolyacrylamide gel. In lane 1 of each Figure are the molecular weightstandards. Lane 2 of each Figure is the purified sCR1[des-A] materialobtained from DUKX-B11 cells. Lane of 3 each is an irrelevant controlmaterial. Lanes 4, 5 and 6 of each gel are sCR1[des-A]sLex at varyingstages during the purification procedure.

FIG. 2A is a coomassie blue stained polyacrylamide gel pattern. Thepredominant bands at approximately 187 kd in lanes 2, and 4-6 are thesCR1[des-A] protein, lane 2 obtained from DUKX-B11 cells and lanes 4-6obtained from LEC-11 cells. FIG. 2B is the same gel as FIG. 2 Westernblotted and probed with and anti-sCR1[des-A] polyclonal serum. Asexpected, all lanes containing sCR1-[des-A], whether derived fromDUKX-B11 cells or LEC-11 cells are positive for sCR1[des-A]. FIG. 2C isthe same blot as FIG. 2B stripped and reprobed with an antibody specificfor the sialyl di-Lewis x antigen represented by the shorthand notationNeuNAcα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc. As expected,only lanes 4-6 containing sCR1[des-A]sLex obtained from LEC-11 cells arepositive for the appropriate carbohydrate structure.

12. EXAMPLE 7

12.1 MONOSACCHARIDE COMPOSITION OF SCR1[DES-A] IS CONSISTANT WITHSLE^(x) GLYCOSYLATION

The monosaccharide composition of the glycans comprising thecarbohydrate moiety of the sCR1[des-A]sLex were analyzed usinggas-liquid chromatography (GLC) following the procedure of Reinhold, V.(1972), Methods in Enzymology, 25:244-249. The conditions of cleavage,derivitization and GLC provided for a quantitative determination of themonocaccarides comprising the the glycoproteins sCR1[des-A] andsCR1[des-A]sLex.

The CHO line utilized for the expression of sCR1 and sCR1[desLHR-A], CHODUKX-B11, lacks α(1,3)fucosyl transferase activity and is thus incapableof sLex glycosylation. Stanley and colleagues have generated a mutantCHO cell line, LEC11, which transcribes endogenous alpha(1-3)fucosyltransferases and can synthesize carbohydrates with fucosylatedterminal structures, including sLe^(x).

In this example, LEC11 cells capable of sLe^(x) glycosylation have beentransfected with the plasmid encoding sCR1[desLHR-A] to producesCR1[desA]sLe^(x). The sCR1[des-A]sLe^(x) was compared to sCR1[des-A]produced in DUKX-B11 cells. When the two glycoproteins where analyzedfor monosaccharide composition the results presented in Table I belowwere obtained:

TABLE I sCR1[des-A] sCR1[des-A]sLex % Wt MR % Wt MR Fuc 1.4 1.1 2.7 1.9Man 4.1 3.0 4.7 3.0 Gal 3.6 2.6 4.7 3.0 GlcN 7.5 4.4 8.9 4.6 NA 2.7 1.14.5 1.7 The values presented in Table I for % Wt are the percentage ofthe particular sugar relative to the total protein. The MR is the molarratio normalized to mannose. Fuc = fucose, Man = mannose, Gal =galactose, GlcN = N-acetyl-glucosamine, NA = sialic acid.

It can be concluded from Table I that the fucose and sialic acid contentof the sCR1[des-A]sLex are consistent with the expectation that the Lec11 cell line is adding the appropriate carbohydrates necessary for thesialylated Lewis x antigen as well as the sialylated di-Lewis x antigen.

sLe^(x) is the carbohydrate ligand for both P- and E-selectin andthereby mediates leukocyte adherence at vascular sites of inflammation.sCR1[desA]sLe^(x) thus combines the anti-inflammatory potential of botha complement regulatory protein and an adhesion molecule.

13. EXAMPLE 8

13.1 MASS SPECTROSCOPY OF THE OLIGOSACCHARIDES FROM SCR1[DES-A]SLEX ISCONSISTANT WITH SLEX GLYCOSYLATION

Mass spectroscopy confirmed the presence of fucose and sialic acidcontaining carbohydrates consistent with sLex glycosylation. Electronmicrospray (Fenn et al., (1989) Science, 246:64-71) followed by massspectroscopy provided an assessment of the N-acetylneuraminic acidgroups (sialic acid), fucosylation, and partial insight into antennaextensions and branching. In this Example sCR1[des-A]sLex was endo-Hdeglycosylated and an ES-MS “fingerprint” was obtained and compared to asimilar “fingerprint” obtained from an endo-H deglycosylated sCR1[des-A]glycoprotein.

Carbohydrates were grouped into bi- tri- and tetra antennary structureseach having the typical trimannose core structure.

In the resulting ion profile each ion was accounted for by reference tocomposition-mass tables compiled for each monosaccharide. The ion m/z1062.8 found in the sCR1[des-A]sLex “fingerprint”, for instance,represents a biantennary structure consistant with a preferred sLexcarbohydrate moiety and can be accounted for as fucosylated sialylatedLewis x antigen of the following structure:

The corresponding structure with equivalent m/z is absent in the“fingerprint” analysis of sCR1[des-A].

The data in Table II represents the percent mole ratio of each of theparticular carbohydrate structures present in the sCR1[des-A] andsCR1[des-A]sLex compostions.

TABLE II Glycoform* sCR1 sCR1[des-A] sCR1[des-A]sLex BiNA₀ 49.6 31.9 8.4BiNA₀F₁ — — 3.6 BiNA₀F₂ — — 1.7 BiNA₁ 19.6 27.0 15.8 BiNA₁F₁ — — 10.9BiNA₁F₂ — — 4.6 BiNA₂ 4.0 13.0 9.0 BiNA₂F₁ — — 9.6 BiNA₂F₂ — — 4.8TriNA₀ 7.4 5.4 1.6 TriNA₀F₁ — — <1 TriNA₀F₂ — — <1 TriNA₁ 4.0 3.8 4.1TriNA₁F₁ — — 1.9 TriNA₁F₂ — — <1 TriNA₂ 1.7 2.7 4.3 TriNA₂F₁ — — 3.5TriNA₂F₂ — — <1 TriNA₃ 1.1 2.7 2.7 TriNA₃F₁ — — 2.0 TriNA₃F₂ — — <1TetraNA₀ 1.8 1.3 — TetraNA₁ ^(#) 1.8 1.6 1.2 TetraNA₂ ^(#) <1 1.3 1.9TetraNA₃ ^(#) <1 <1 1.6 TetraNA₄ ^(#) <1 <1 1.3 BiNA₀-(Gal) 5.5 4.8 1.9BiNA₁-(Gal) <1 1.8 <1 BiNA₀-(Fuc) 1.7 1.5 <1 *core fucosylated; approx.3% are non-fucosylated #including sialyl lewis(x) in TP18

ES-MS analisys of sCR1[desA]sLe^(x)-derived oligossaccharide structuresis consistent with sLe^(x) glycosylation.

14. EXAMPLE 9

14.1 FUNCTIONAL ACTIVITY OF sCR1[des-A]sLex IN VITRO

In this Example the functional activities of the purified proteinssCR1[des-A] and sCR1[des-A]sLex are compared in vitro. sCR1[desA]sLe^(x)inhibited E-selectin mediated binding of U937 cells to activated humanaortic endothelial cells in a concentration-dependent manner in vitro.In this static adhesion assay, sCR1[desA]sLe^(x) inhibited binding ofU937 cells by 50% at a final concentration of 250 ug/ml.

14.1.1 Static Adhesion Blocking Assay.

Aortic endothelial cells (Clonetics), confluent in 96-well microtiterplates, were stimulated with TNF (100 U/ml) for 4 h at 37° C. The cellswere then washed twice with DNEN supplemented with 1% FBS. Serialdilutions of sCR1[desA]sLe^(x) and sCR1[desLHR-A] were made to achievefinal concentrations of 500, 250, 125, 62.5, and 0 ug/ml. To each well,5×10⁵ U937 (obtained from ATCC) cells in 20 μl of DMEM were added andincubated for 20 min at 37° C. The wells were filled with media, sealed,and centrifuged inverted at low speed (150×g) for 5 min. The seal wasremoved, the plates blotted, and the number of bound cells in threemicroscope fields was determined.

14.2 RESULTS

Using this in vitro static adhesion assay, sCR1[desA]sLe^(x) inhibitedE-selectin mediated adhesion in a concentration-dependent manner. Humanaortic endothelial cells were stimulated with TNF to induce cell surfaceexpression of E-selectin. Surface expression of E-selectin wasdetermined using DREG-56 (a monoclonal antibody specific for E-selectin)in an immunocytochemical staining protocol. U937 cells, shown to havesurface sLe^(x) by flow cytometric analysis with CSLEX-1, were shown toadhere to the activated endothelial cells. The adherence phenomenonbetween the activated aortic endothelial cells and U937 cells was shownto require the presence of calcium, a hallmark of selectin-mediatedadhesion. The E-selectin dependent adhesion of U937 cells to activatedendothelial cells was inhibited by sCR1[desA]sLE^(x) in a concentrationdependent manner.

FIG. 3 details the results of this experiment. The black bars representthe sCR1[des-A] material obtained from DUKX-B11 cells. The bars withhorizontal lines represent sCR1[des-A]sLex material obtained form LEC-11cells. The sCR1[des-A]sLex material inhibited binding of U937 cells toactivated aortic endothelial cells in a concentration dependent manner.

15. EXAMPLE 10

15.1 IN VIVO FUNCTIONAL ACTIVITY OF sCR1[des-A]sLex

Endothelial upregulation of selecting, to which oligosaccharides such assialyl Lewis^(x), and sialyl di-Lex bind, are important adhesionpromoting molecules for neutrophils. The soluble complement receptor 1(sCR1), which is a potent inhibitor of complement, has been expressed ina truncated form, with and without decoration with SLe^(x)(sCR1[desA]sLe^(x) and sCR1[desA], respectively). Both compounds havesubstantial complement-blocking activity in vitro as demonstrated above.In a rat model of P-selectin-dependent acute lung injury, the rank orderof protective activity for these inhibitors is:sCR1[desA]sLe^(x)>sCR1≧sCR1[desA]. By taking advantage ofoligosaccharide decoration of sCR1[desA] to cause binding to theactivated endothelium at sites of selectin expression, the complementinhibitor can be “targeted” to an inflammatory site.

The inhibitor preparations were employed in vivo in the CVF model of ratlung injury. Four separate groups of rats (n=5 each) were pretreatedintravenously with 0.4 ml sterile saline, sCR1, sCR1[desA] orsCR1[desA]sLe^(x) (each at 15 mg/kg body weight) and injectedintravenously 5 min before intravenous infusion of CVF. Also, a negativecontrol group (infused with sterile saline in the absence of CVF) wasemployed (n=5). Thirty min. after infusion of CVF or sterile saline,animals were killed with an overdose of ketamine and 1.0 ml bloodobtained from the inferior vena cava (a).

As shown by the data in FIG. 4, treatment of animals with sCR1[desA],sCR1[desA]sLe^(x) or sCR1 reduced (as a percentage) MPO content in lungby 40±3, 64±3 and 55±4, respectively (FIG. 4C). FIG. 4C is a measure ofthe accumulation of neutrophils in the lung as estimated by measurementof myeloperoxidase activity (MPO). When compared statistically,sCR1[desA]sLe^(x) and sCR1 were more effective than sCR1[desA] inreducing MPO content.

FIG. 4B also describes the protective effects of sCR1, sCR1[des-A], andsCR1[des-A]sLex from hemorrhagic lung injury induced by CVF. FIG. 4B isthe measurement of the reduction over control of hemorrhage as measuredby a radiolabelled red blood cell leakage into the lung from the bloodvessel. sCR1[des-A]sLex reduced hemorrhage approximately 65 percent overcontrol. Permeability is a measure of radiolabelled protein leakage fromthe blood vessels of the lung. sCR1[des-A] reduced permeablityapproximately 60 5 over control in this experiment. Thus,sCR1[desA]sLe^(x) appears to be the most effective of the threecomplement inhibitors in reducing injury in this inflammatory model.

At the time of sacrifice (30 min after intravenous infusion of CVF),plasma was obtained and evaluated for the concentration of sCR1[desA]antigen using an ELISA sandwich technique. Plasma from CVF infusedanimals that were otherwise untreated revealed <50 ng/ml measurablesCR1[desA] antigen, while plasma antigenic levels of sCR1[desA] in thesCR1[desA] and sCR1[desA]sLe^(x) treated animals (injected with CVF)were 267±28.2 and 154±33.9 μg/ml, respectively. These data would beconsistent with an accelerated selectin-dependent removal ofsCR1[desA]sLe^(x) from the vascular compartment.

These data demonstrate that, in the P-selectin-dependent model of acutelung injury occurring after CVF-induced systemic activation ofcomplement, the complement inhibitor, sCR1[desA] decorated with sLexgroups provides the most effective protection (as compared to sCR1[desA]or sCR1) in this model of neutrophil-dependent injury. Reduced MPOcontent in lung suggests that sCR1[desA]sLe^(x) more effectively blockedP-selectin-dependent adhesion of neutrophils to the activatedendothelium, which is known to be upregulated for P-selectin. Each ofthree complement inhibitors had protective effects that were associatedwith diminished buildup of lung MPO.

By reducing the amount of endothelial activation (upregulation ofP-selectin) and diminishing neutrophil activation (resulting ingeneration of toxic oxygen products), complement blockage interfereswith injury-promoting interactions between neutrophils and theendothelium. In this model of lung injury it is known that bothneutrophils and toxic oxygen products are required for full developmentof injury. The close proximity between neutrophils and the endotheliumis required for the most effective action of toxic oxygen products (fromneutrophils) on the endothelium. These adhesive interactions can beblocked with antibodies to P-selectin or leukocytic β2 integrins, ox byinfusion of sLe^(x). In all cases the protective effects of theseinterventions are associated with diminished levels of tissue MPO. Theenhanced inhibitory activity of sCR1[desA]sLe^(x) would be consistentwith the interpretation that, as CVF-induced complement activationoccurs (thus causing endothelial upregulation of P-selectin),sCR1[desA]sLe^(x) can selectively bind to endothelial P-selectin,providing localized protection against further complement activation.Localization of sCR1[desA]sLe^(x) to areas of activated endothelium issupported by the immunostaining data and could also explain why residualplasma levels of sCR1[desA] antigen at 30 min were nearly 50% lower insCR1[desA]sLe^(x) treated animals than those treated with sCR1[desA].

The ability to “target” complement inhibitors to the endothelium basedon the ability of sLex to cause binding of sCR1[desA]sLe^(x) toP-selectin (or to E-selectin) provides a unique strategy to optimize theprotective effects of these inhibitors. Since ischemia-reperfusioninjury to the myocardium appears to be P-selectin-dependent, it ispossible that in humans treatment of ischemia-reperfusion injury wouldbenefit from the use of such inhibitors, as well as other conditions inwhich selectin and complement activation molecules participate inoutcomes leading to injury.

16. EXAMPLE 11

16.1 GENERATION OF A SOLUBLE COMPLEMENT RECEPTOR TYPE 1 WITH SELECTINBINDING ACTIVITY

We describe herein another soluble form of complement receptor type 1with selectin binding activity. This bifunctional molecule is a valuabletool in modulating the inflammatory response.

16.1.1. CELL LINES

The cell line K562 was supplied by Dr. Lloyd Klickstein, Center forBlood Research, 200 Longwood Avenue, Boston, Mass. 02115, and isgenerally available for the American Type Culture collection (Rockville,Md.). HL-60 cells were obtained from the ATCC.

16.1.2. MONOCLONAL ANTIBODIES

Rabbit polyclonal antiserum specific for CR1 can be obtained by standardtechniques known in the art by immunizing rabbits with human complementreceptor type 1. Monoclonal antibodies to CD15 are commerciallyavailable and can be obtained from Dako, California and for instance,clone 28 may be obtained from AMAC, Inc., Maine. Murine monoclonalantibody 3C6. D1l was obtained from a standard fusion using the methodoriginally described by Kohler and Milstein (1975, Nature 256:495-497).Balb/c mice were immunized at 3-4 week intervals with purifiedrecombinant complement receptor type 1 i.p. in Freunds adjuvant. Fourweeks after the third immunization, mice were boosted intravenously with10 μg CR1 and the spleen was removed four days later. Spleenocytes werefused with NSO myeloma cells by addition of 1 ml of 50% PEG-1500(Boehringer Mannheim, Indianapolis Ind.), then diluted with 20 ml ofOPTI-MEM media (GIBCO). After fusion, the cells were plated into wellsof 96 well flat bottomed plates and selected in medium containing HAT(GIBCO). Wells positive for growth were screened for the production ofanti CR1 mAbs using a CR1 capture antibody. Control antibodies weremurine IgM and murine IGg1, commercially available from BectonDickinson, Franklin Lakes, N.J., and Tago, Calif.

16.1.3. TRANSFECTION

K562 cells expressing complement receptor type 1 (CR1) can be obtainedby transfecting host cells by electroporation with full length CR1obtained from construct piABCD (Klickstein, et al., 1988, 168:1699).Approximately five million K562 cells suspended in 0.8 ml medium aremixed with approximately 20 μg plasmid DNA, linearized with SpeJ, andsubjected to 200 volts, 960 μF using a genepulser electroporationapparatus (BioRad). After several days in culture cells expressing thesoluble CR1 gene product can be selected for the expression of solubleCR1 using the CELLFREE® CD35 Bead Assay Kit obtained from T CellDiagnostics, Inc. Cambridge, Mass.

Alternatively, the calcium phosphate-mediated transfection of K562 cellscan be accomplished using the method of Graham and van der Erb (1973)Virology 52:456-467.

16.1.4. CELL LYSATES

Cell lines transfected to express the appropriate molecules or cell lineendogenously expressing the appropriate molecules were solubilized at5×107 cells/ml in lysis buffer containing 10 mM Tris pH 8.0, 1% nonidetP-40 (NP-40), 10 mM iodoacetamide (IAA), 1 mM phenylmethly sulfonylfluoride (PMSF), 0.04% aprotinin and 0.3 mM N-tosyl-L-phenylalaninechloromethyl ketone (TPCK).

16.1.5. WESTERN BLOT ANALYSIS

The reactivity of CR1 purified by affinity chromatography from K562supernatants was tested by Western blot analysis. Supernatants from K562transfected with the full length CR1 were fractionated by 4-20% SDS-PAGEand then transferred to nitrocellulose sheets. The sheets were firstblocked with blocking buffer (1% bovine serum albumin inphosphate-buffered saline in PBS). After blocking, the sheets wereincubated with either antibody 3C6. D11 about 2-3 μg/ml(anti-CR1),anti-CD15 (about 20 μg/ml, or irrelevant isotype matched controlantibody C305 (IgM, about 20 μg/ml), or W112 (IgG, about 2-5 μg/ml).After 1-2 hour incubation in the presence of the primary antibodies thesheets were washed with a solution of PBS and 0.05% Tween-20. Afterwashing the sheets were incubated with horseradish peroxidase (HRP)conjugated goat anti-mouse antibody. After washing, color was developedwith an HRP substrate,

16.2. RESULTS

As expected, the material recovered from the K562 cell culturesupernatants can be detected by Western blot using antibodies to theLewis X antigen as well as monoclonal anti-CR1 antibodies.

16.3. PHYSICAL CHARACTERIZATION OF KCR1

To define the specific carbohydrate structures of the KCR1 recoveredsupra both affinity purified KCR1 and neuraminidase treated KCR1 weretested for their ability to bind anti-CR1 immobilized on wells of 96well plates. Detection was with an anti-CD15 antibody which is reactivewith the Lex SLe^(x) ligand structures. Treatment of the KCR1 withneuraminidase removes terminal sialic acid residues from the SLe^(x)oligosaccharide structures yielding the Lewis X structure. Results ofthis analysis are presented in Table III.

TABLE III Reactivity of KCR1 with anti-CD15 Antibody Test Sample Bound(OD₄₉₀₋₆₅₀) Monoclonal Antibody CR1¹ 0.059 ± 0.002 KCR1² 0.135 ± 0.001nKCR1³ 0.130 ± 0.006 KCR1⁴ 0.110 ± 0.024

All samples are the mean plus or minus the standard deviation of themean for four samples excluding the control which is the average of twosamples.

¹ CR1 represents a sample of CR1 obtained from Chinese Hamster ovarycells which does not contain appropriate carbohydrate structures forbinding the CD15 antibody.

² KCR1 represents a sample of affinity purified CR1 produced in K562cells. The sample was concentrated by a Centricon™ concentrator prior toassay

³ nKCR1 represents a sample of CR1 produced in K562 cells which wastreated with neuraminidase. Neuraminidase treatment consisted ofincubating the sample in the presence of neuraminidase prior to assay.

⁴ KCR1 represents a sample of CR1 affinity purified from K562 cells anduntreated prior to assay.

17. GENERALIZED ASSAY FORMATS TO DETECT FUNCTIONAL ACTIVITY

The compositions of the invention may also be evaluated for theirability to block intercellular adhesion to certain cells, for instance,activated endothelial cells thereby inhibiting a primary event in theinflammatory response. This evaluation may be achieved by a number ofmethods; the following methods being described as specific proceduresthat were employed in this regard or that may be useful in additionthereto:

A. COMPETITIVE INHIBITION OF HL-60 BINDING TO E AND P SELECTINS

a) cells expressing E or P selectin(activated platelets or cellstransfected with and expressing selectins on their surface, refLarsen,et al.) are grown in 96-well microtiter plates to confluence.

b) HL-60 cells are added at 4 deg Centigrade in the presence or absenceof CR1 or CR1 analogues and allow to settle and bind for 30 min.

c) non-adherent cells are removed by inverting the plates andcentrifuging at 150 xg for 5 min.

d) the plates are scored for the number of bound HL-60 cells permicroscope field.

B. IN VIVO ASSAY FOR SELECTIN BINDING

a) induce P-selectin up-regulation in rats with CVF in accordance withthe method of Mulligan et al., 1992, J. Clin. Invest. 90: 1600-1607 .

b) inject radio-labelled sLEX-CR1 chimera or analogues vs TP10 anddetermine distribution of radiolabel.

C. IN VIVO ASSAY FOR COMPOSITION EFFICACY

Mulligan et al.(“Role of Leukocyte Adhesion Molecules inComplement-Induced Lung Injury”, J. Immunol. Vol. 150, 2401-24061, No.6, Mar. 15, 1993,) describe the role of P selectin in lung vascularendothium injury in rats after cobra venom factor (CVF) activation ofcomplement. Since it has previously been shown that complement has aprotective effect in preventing acute microvascular injury of the lunginduced by CVF, it is desirable to home the CR1 to the site of theinjury via the selectin ligand. In order to assess the localization ofthe complement, twenty units of CVF per kg body weight is injectedintravenously into male 300-350 gram Long Evans rats. To assess thelocalization of the CR1 to the lung ¹²⁵I-CR1-sLe^(x) (approximately 500μCi), or in control animals ¹²⁵I-CR1 is injected at for instance 15mg/kg body weight. Since CVF induced lung injury is instantaneous,localization can be assessed by assessing tissue incorporation ofradiolabelled CR1-sLe^(x) by standard techniques approximately 30minutes after injection.

The CVF model can also be used to assess the ability of the sLextoprevent the primary events in inflammation such as neutrophilsequestration and subsequent rolling and firm attachment. See also,Mulligan et. al., Role Endothelial-Leukocyte Adhesion Molecule 1(ELAM-1) in Neutropnil-Mediated Lung Injury in Rats, J. Clin. Invest.,Vol. 88, October 1991, 1396-1406, and Mulligan et al.,“Neutrophil-dependent Acute Lung Injury,” J. Clin. Invest., Vol. 90,October 1992, 1600-1607.

18. OTHER TECHNIQUES FOR PREPARATIONS OF COMPOSITIONS

18.1. MUTAGENESIS

CHO (Chinese hamster ovarian) cells that express sCR1 are used. Thecells in suspension (at about 2×10⁵ cells/ml) can be incubated for 18hrs with EMS, washed, and relative plating efficiencies determined.Mutagenized cells may be cultured for seven days to allow expression ofacquired mutations. Cells can be aliquoted at about 10⁶ cells/100-mmtissue culture dish in medium containing about 10% fetal calf serum andthe appropriate concentration of the primary selective lectin(s). Aftersix days, the plates are washed twice with alpha medium and the scondaryselective lectin(s) added in alpha medium containing 10% fetal calfserum. After approximately four more days of incubation, the largestcolonies are picked into alpha medium containing 10% fetal calf serumand the plates stained with 2% methylene blue in 50% methanol. Controlplates which contained no lectin or only the primary selective lectin(s)are stained after 8 days and relative plating efficiencies determined.

18.2. CELL FUSION

Approximately 1×10⁸ cells expressing the α1,3-fucosyl transferase and5×10⁷ cells expressing the desired complement protein which have beenpreviously suspended in DMEM, were pelleted by centrifugation at 200×gfor 5 minutes and warmed to 37° C. The pellet was resuspended in 1 ml ofculture medium containing about 1 g/ml of polyethylene glycol (PEG)4000, supplemented with 5% DMSO at 37° C. with gentle mixing. The cellswee then spun at 100×g for 2 minutes, 4.5 ml of supplemented medium wasadded over the next 3 minutes followed by 5 ml of supplemented mediumover the next two minutes. Then the tube was filled with supplementedmedium. As is well known in the art, timing of these steps is important.

The cells were pelleted by centrifugation at 100×g for 5 min at roomtemperature, then the supernatant was aspirated. The cell pellet wasresuspended in medium, but care was taken not to force the dispersion ofsmall cell clumps. The cells were plated in a 96-well plate in limitingdilution (To ensure growth, the wells of the plate may contain feedercells). Culture medium containing mycophenolic acid was added, and thenreplaced as often was deemed necessary to ensure cell selection. Cellsexpressing sLex or sialyl di-Lex are then selected.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

11 68 base pairs nucleic acid single linear DNA unknown 1 CGCCCGGTCTCCCCTTCTGC TGCGGAGGAT 30 CCCTGCTGGC GGTTGTGGTG CTGCTTGCGG 60 TGCCGGTG 6872 base pairs nucleic acid single linear DNA unknown 2 CCAGGCCACCGGCAGCGCAA GCAGCACCAC 30 AACCGCCAGC AGGGATCCTC CGCAGCAGAA 60 GGGGAGACCGGG 72 66 base pairs nucleic acid single linear DNA unknown 3 GCCTGGGGTCAATGTCAAGC CCCAGATCAT 30 TTTCTGTTTG CCAAGTTGAA AACCCAAACC 60 AATGCA 6656 base pairs nucleic acid single linear DNA unknown 4 TTGGTTTGGGTTTTCAACTT GGCAAACAGA 30 AAATGATCTG GGGCTTGACA TTGACC 56 31 base pairsnucleic acid single linear DNA unknown 5 CCCCCCCTCG AGGTCGACGGTATCGATAAG 30 C 31 56 base pairs nucleic acid single linear DNA unknown6 TATCAAATGC GGCCGCTAAG AATACCCTAG 30 ATCTGGAGCA GCTTGGTAAC TCTGGC 56 19base pairs nucleic acid single linear DNA unknown 7 GATGAACTAG TCTCGAGAG19 19 base pairs nucleic acid single linear DNA unknown 8 CTCTCGAGACTAGTTCATC 19 40 base pairs nucleic acid single linear DNA unknown 9CGACACTTGA AAGACAAGCG CTACCAGTGA 30 CATTTTGGGG 40 13 base pairs nucleicacid single linear DNA unknown 10 CGCTTAAGCT CGA 13 17 base pairsnucleic acid single linear DNA unknown 11 GATCTCGAGC TTAAGCG 17

What is claimed is:
 1. A pharmaceutical composition comprising a solublecomplement receptor 1 polypeptide selected from the group consisting ofsCR1 and sCR1 lacking LHR-A bound to a carbohydrate structure which is aselectin ligand in admixture with a pharmaceutically acceptable carrier,wherein said soluble complement receptor 1 polypeptide inhibits aprimary event in the inflammatory response.
 2. The pharmaceuticalcomposition of claim 1 wherein said selectin ligand is a Lewis Xantigen.
 3. The pharmaceutical composition of claim 2 wherein saidselectin ligand is a sialyl Lewis X antigen.
 4. The pharmaceuticalcomposition of claim 1 wherein the soluble complement receptor 1polypeptide blocks intercellular adhesion.
 5. The pharmaceuticalcomposition of claim 1 wherein the soluble complement receptor 1polypeptide binds a cellular adhesion molecule.
 6. The pharmaceuticalcomposition of claim 1 wherein the soluble complement receptor 1polypeptide binds to activated endothelial cells.
 7. A conjugatecomprising at least one soluble complement receptor type 1 (CR1)polypeptide selected from the group consisting of sCR1 and sCR1 lackinglong homologous repeat-A (LHR-A) bound to at least one carbohydratestructure which is a selectin ligand which conjugate inhibits a primaryevent in the inflammatory response.
 8. The conjugate of claim 7 in whichthe conjugate blocks intercellular adhesion.
 9. The conjugate of claim 7in which the conjugate binds to a cellular adhesion molecule.
 10. Theconjugate of claim 7 in which the conjugate binds to activated end ofendothelia cells.
 11. The conjugate of claim 7 in which the selectinligand is selected from the group consisting of ligand of E-selectinP-selectins, and L-selectins.
 12. The conjugate of claim 7 in which theconjugate binds to an E-selectin.
 13. The conjugate of claim 7 in whichthe conjugate binds to a P-selectin.
 14. The conjugate of claim 7 inwhich the conjugate binds to an L-selectin.
 15. The conjugate of claim 7in which said conjugate binds to a lectin domain within said selectinand has a binding affinity of at least about 10⁴M⁻¹.
 16. The conjugateof claim 7 in which said at least one carbohydrate is an N-linkedcarbohydrate.
 17. The conjugate of claim 16 in which the N-linkedcarbohydrate is fucosylated.
 18. The conjugate of claim 16 in which thecarbohydrate structure comprises an oligosaccharide related to the LewisX carbohydrate.
 19. The conjugate of claim 18 in which the carbohydratecomprises the Lewis X carbohydrate.
 20. The conjugate of claim 17 inwhich the N-linked carbohydrate is sialylated.
 21. The conjugate ofclaim 20 in which the carbohydrate structure comprises the sialyl LewisX carbohydrate.
 22. The conjugate of claim 7 in which the solublecomplement receptor polypeptide comprises at least LHR B, LHR C, and LHRD up to and including the first alanine residue of the transmembraneregion of complement receptor type
 1. 23. A soluble complementinhibitory protein comprising a soluble CR1 polypeptide selected fromthe group consisting of sCR1 and sCR1 lacking LHR-A, which polypeptidebears a carbohydrate structure which is a selectin ligand, wherein saidsoluble complement inhibitory protein inhibits a primary event in theinflammatory response.
 24. The complement inhibitory protein of claim 23in which the selectin ligand is a Lewis X antigen.
 25. The complementinhibitory protein claim 24 in which the Lewis X antigen is a sialylLewis X antigen.
 26. The soluble complement inhibitory protein of claim23 wherein the protein blocks intercellular adhesion.
 27. The solublecomplement inhibitory protein of claim 23 wherein the protein binds acellular adhesion molecule.
 28. The soluble complement inhibitoryprotein of claim 23 wherein the protein binds to activated endothelialcells.